Peptide-based multimeric targeted contrast agents

ABSTRACT

Peptides and peptide-targeted multimeric contrast agents are described, as well as methods of making and using the contrast agents.

RELATED APPLICATION DATA

This application is a divisional of U.S. application Ser. No.10/209,183, filed Jul. 30, 2002, now abandoned which claims priority toU.S. Provisional Application Ser. No. 60/308,721, filed Jul. 30, 2001,and is related to co-pending U.S. application Ser. No. 10/209,172, filedJul. 30, 2002, and to co-pending U.S. application Ser. No. 10/786,791,filed Feb. 25, 2004, all of which are incorporated herein by referencein their entirety.

TECHNICAL FIELD

This invention relates to contrast agents for diagnostic imaging, andmore particularly to peptide-targeted, multimeric contrast agents,wherein a peptide functions as a targeting group and a point ofattachment for one or more chelates at both the amino and carboxytermini of the peptide.

BACKGROUND

Diagnostic imaging techniques, such as magnetic resonance imaging (MRI),X-ray, nuclear radiopharmaceutical imaging, ultraviolet-visible-infraredlight imaging, and ultrasound, have been used in medical diagnosis for anumber of years. Contrast media additionally have been used to improveor increase the resolution of the image or to provide specificdiagnostic information.

To be effective, the contrast media must interfere with the wavelengthof electromagnetic radiation used in the imaging technique, alter thephysical properties of tissue to yield an altered signal, or, as in thecase of radiopharmaceuticals, provide the source of radiation itself.MRI and optical methods are unique among imaging modalities in that theyyield complex signals that are sensitive to the chemical environment.While the signal from X-ray or radionuclide agents remains the samewhether agents are free in plasma, bound to proteins or other targets,or trapped inside bone, certain contrast agents for MRI and opticalimaging will have different signal characteristics in differingphysiological environments. It is important that the contrast agent besufficiently sensitive and present at high enough concentration so thatsignal changes can be observed.

Complexes between gadolinium or other paramagnetic ions and organicligands are widely used to enhance and improve MRI contrast. Gadoliniumcomplexes increase contrast by increasing the nuclear magneticrelaxation rates of protons found in the water molecules that areaccessible to the contrast agents during MRI (Caravan, P., et al., R. B.Chem. Rev. 99, 2293 (1999)). The relaxation rate of the protons in thesewater molecules increases relative to protons in other water moleculesthat are not accessible to the contrast agent. This change in relaxationrate leads to improved contrast of the images. In addition, thisincrease in relaxivity within a specific population of water moleculeprotons can result in an ability to collect more image data in a givenamount of time. This in turn results in an improved signal to noiseratio.

Imaging may also be performed using light, in which case an optical dyeis chosen to provide signal. In particular, light in the 600-1300 nm(visible to near-infrared) range passes relatively easily throughbiological tissues and can be used for imaging purposes. The light thatis transmitted through, or scattered by, reflected, or re-emitted(fluorescence), is detected and an image generated. Changes in theabsorbance, reflectance, or fluorescence characteristics of a dye,including an increase or decrease in the number of absorbance peaks or achange in their wavelength maxima, may occur upon binding to abiological target, thus providing additional tissue contrast. In somesituations, for example the diagnosis of disease close to the bodysurface, UV or visible light may also be used.

A need persists for contrast agents that can deliver sufficientconcentrations of the imaging moiety to the target to improve thesensitivity of the imaging process as well as contrast agents that havea sufficient half-life in vivo.

SUMMARY

The invention is based on peptides and peptide-targeted multimericcontrast agents for MR, optical, and radionuclide imaging, wherein asingle peptide can function both as a targeting group and a point ofattachment for one or more chelates at both the N- and C-termini, eitherdirectly or via an optional intervening linker. Surprisingly, contrastagents of the invention maintain binding affinity for biological targetssuch as fibrin and high relaxivity. Agents of the invention have asufficient half-life following in vivo administration such thateffective imaging studies can be performed.

In one aspect, the invention features purified peptides that include theamino acid sequence: P*—Y*—X₁*-L* (SEQ ID NO:1), wherein P* is a prolineor a non-natural derivative thereof; Y* is a tyrosine or a non-naturalderivative thereof; X₁* is G or D or a non-natural derivative of G or D;L* is a leucine or a non-natural derivative thereof; and wherein atleast one of P*, Y*, X₁*, and L* is a non-natural derivative of therespective amino acid. X₁* can be G or D and L* can be leucine. In someembodiments, P* is proline or 4-hydroxyproline, and Y* is tyrosine or anon-natural derivative of tyrosine substituted at the 3 position with amoiety selected from the group consisting of F, Cl, Br, I, and NO₂.Compounds of the invention can include such peptides linked to athrombolytic agent.

In another aspect, the invention features purified peptides that includethe amino acid sequence X₁—X₂—C—P*—Y—X₃-L-C—X₄—X₅—X₆ (SEQ ID NO:2),wherein: P* is a proline or a non-natural derivative thereof; Y* is atyrosine or a non-natural derivative thereof; X₁ is selected from thegroup consisting of W, Y, F, S, Bip, Hx, Dpr, Cy, Gu, Ad, Hfe, 3-Pal,4-Pal, DopaMe2, nTyr, dW, dF, F(3/4*), and Y(3*), wherein F(3/4*) is aphenylalanine substituted at either the 3 or the 4 position with amoiety selected from the group consisting of CH₃, CF₃, NH₂, CH₂NH₂, CN,F, Cl, Br, I, Et, and OMe, and wherein Y(3*) is a tyrosine substitutedat the 3 position with a moiety selected from the group consisting of F,Cl, Br, I, and NO₂; X₂ is selected from the group consisting of E, H,dE, S, H(Bzl), 2-Pal, Dpr, and Th; X₃ is selected from the groupconsisting of G and D; X₄ is selected from the group consisting of H, F,Y, and W; X₅ is selected from the group consisting of I, L, V, N, Bpa,Bal, Hfe, Nle, Tle, Nval, Phg, Cha, Tai, Fua, Th, 4-Pal, and F(3/4*),wherein F(3/4*) is a phenylalanine substituted at either the 3 or the 4position with a moiety selected from the group consisting of CF₃, Et,iPr, and OMe; X₆ is selected from the group consisting of N, Q, I, L,and V, or X₆ is not present; and wherein at least one of X₁, X₂, X₅, P*,and Y* is a non-natural derivative of an amino acid. For example, P* canbe proline or 4-hydroxyproline, and Y* can be tyrosine or a non-naturalderivative of tyrosine substituted at the 3 position with a moietyselected from the group consisting of F, Cl, Br, I, and NO₂. Thepurified peptides can be capable of forming a disulfide bond undernon-reducing conditions and can have specific binding affinity forfibrin. In some embodiments, the peptides include a disulfide bond.Compounds of the invention can include such peptides linked to athrombolytic agent.

The invention also features purified peptides having an amino acidsequence selected from the group consisting ofW-dE-C—P(4-OH)—Y(3-Cl)-G-L-C—W—I-Q (SEQ ID NO:4), Y-dE-C—P(4-OH)—Y(3-Cl)-G-L-C—Y—I-Q (SEQ ID NO:5),Y-dE-C—P(4-OH)—Y(3-Cl)-G-L-C—W—I-Q (SEQ ID NO:6),W-dE-C—P(4-OH)—Y(3-Cl)-G-L-C—Y—I-Q (SEQ ID NO:7),W-dE-C—P(4-OH)—Y(3-Cl)-D-L-C—W—I-Q (SEQ ID NO:8),Y-dE-C—P(4-OH)—Y(3-Cl)-D-L-C—Y—I-Q (SEQ ID NO:9),Y-dE-C—P(4-OH)—Y(3-Cl)-D-L-C—W—I-Q (SEQ ID NO:10),W-dE-C—P(4-OH)—Y(3-Cl)-D-L-C—Y—I-Q (SEQ ID NO:11),F(4-OMe)—H—C—P(4-OH)—Y(3-Cl)-D-L-C—H-I-L (SEQ ID NO:12),Y—H—C—P(4-OH)—Y(3-Cl)-G-L-C—W—I-Q (SEQ ID NO:13),W-dE-C—P—Y(3-Cl)-G-L-C—W—I-Q (SEQ ID NO:14),W-dE-C—P(4-OH)—Y-G-L-C—W—I-Q (SEQ ID NO:15), andF—H—C—P-(4-OH)—Y(3-Cl)-D-L-C—H—I-L (SEQ ID NO:16). The peptides can becapable of forming a disulfide bond under non-reducing conditions, andin some embodiments, the peptides include a disulfide bond. The peptidescan have specific binding affinity for fibrin. Compounds of theinvention can include such peptides linked to a thrombolytic agent.

In some embodiments, P* is proline; Y* is tyrosine; X₁ is selected fromthe group consisting of W, Y, F, S, Bip, Hx, Dpr, Cy, Gu, Ad, Hfe,3-Pal, 4-Pal, DopaMe2, nTyr, dW, dF, F(3/4*), and Y(3*), wherein F3/4*is a phenylalanine substituted at either the 3 or the 4 position with amoiety selected from the group consisting of CH₃, CF₃, NH₂, CH₂NH₂, CN,F, Cl, Br, I, Et, and OMe, and wherein Y3* is a tyrosine substituted atthe 3 position with a moiety selected from the group consisting of F,Cl, Br, I, and NO₂; X₂ is selected from the group consisting of dE,H(Bzl), 2-Pal, Dpr, and Th; X₃ is selected from the group consisting ofG and D; X₄ is selected from the group consisting of H, F, Y, and W; X₅is selected from the group consisting of I, L, V, N, Bpa, Bai, Hfe, Nie,Tle, nVal, Phg, Cha, Taz, Fua, Th, 4-Pal, and F(3/4*), wherein F3/4* isa phenylalanine substituted at either the 3 or the 4 position with amoiety selected from the group consisting of CF₃, Et, iPr, and Ome,wherein at least one of X₁, X₂, or X₅ is a non-natural amino acidderivative; and X₆ is selected from the group consisting of N, Q, I, L,and V, or X₆ is not present. Such peptides can be capable of forming adisulfide bond under non-reducing conditions, and in some embodiments,the peptides include a disulfide bond. The peptides can have specificbinding affinity for fibrin.

In other embodiments, the invention features purified peptides thatinclude the amino acid sequence: C—P*—Y*—X₁-L-C (SEQ ID NO:3), whereinX₁ is G or D, P* is proline or its non-natural derivative4-hydroxyproline; and Y* is tyrosine or a non-natural derivative oftyrosine substituted at the 3 position with a moiety selected from thegroup consisting of F, Cl, Br, I, and NO₂; provided that at least one ofP* or Y* is a non-natural derivative of the respective amino acid. Thepurified peptides can be capable of forming a disulfide bond undernon-reducing conditions and can have specific binding affinity forfibrin. In some embodiments, the peptides include a disulfide bond.Compounds of the invention can include such peptides linked to athrombolytic agent.

The invention also features purified peptides that include the aminoacid sequence: C-D-Y—Y-G-T-C—X₁₀ (SEQ ID. NO:17), wherein X₁₀ isselected from the group consisting of n(decyl)G, n(4-PhBu)G, MeL, Bpa,Bip, Me-Bip, F(4*), F(3-Me), F(3,4-difluoro), Amh, Hfe, Y(3,5-di-iodo),Pff, 1Nal, d1Nal, and MeL, wherein F(4*) is a phenylalanine substitutedat the 4 position with a moiety selected from the group consisting ofEt, CF₃, I, and iPr. Purified peptides can include the amino acidsequence C-D-Y—Y-G-T-C—X₁₀—X₁₁ (SEQ ID. NO:18), wherein X₁, is selectedfrom the group consisting of D, dD, PD, Inp, Nip, Me-D, dC, Cop, andCmp. For example, a peptide can have the follow amino acid sequences:L-P—C-D-Y—Y-G-T-C-n(Decyl)G-dD (SEQ ID NO:19),L-P—C-D-Y—Y-G-T-C-n(Decyl)G-D (SEQ ID NO:20), L-P—C-D-Y—Y-G-T-C-Bip-D(SEQ ID NO:21), L-P—C-D-Y—Y-G-T-C-Bip-dD (SEQ ID NO:22),L-P—C-D-Y—Y-G-T-C-MeL-Inp (SEQ ID NO:23), L-P—C-D-Y—Y-G-T-C-MeL-Cmp (SEQID NO:24), or L-P—C-D-Y—Y-G-T-C-MeBip-D (SEQ ID NO:25). The purifiedpeptides can be capable of forming a disulfide bond under non-reducingconditions and can have specific binding affinity for fibrin. In someembodiments, the peptides include a disulfide bond. Compounds of theinvention can include such peptides linked to a thrombolytic agent.

In another aspect, the invention features a method of making an MRimaging agent. The method includes reacting a peptide having anN-terminal amine functional group with a linker-subunit moiety to form amodified peptide having both a C-terminal amine functional group andN-terminal amine functional group; covalently attaching a linker moietyto the C-terminal amine functional group and to the N-terminal aminefunctional group to form a precursor MR imaging agent; and convertingthe precursor MR imaging agent to the MR imaging agent. Thelinker-subunit moiety can be selected from the group consisting of:

wherein n is an integer from 1 to 4; m is an integer selected 1 to 12;and R is an aliphatic or aromatic group. The linker moiety can beselected from the group consisting of

wherein m is an integer from 1 to 4; n is an integer from 0 to 4; LG isa leaving group; and R′ and R″ independently are selected from the groupconsisting of hydrogen and a chemical protecting group.

The linker moiety also can be selected from the group consisting of:

wherein LG is a leaving group; and R¹ and R² independently are selectedfrom the group consisting of hydrogen and a chemical protecting group.The LG can be selected from the group consisting of —OH, activatedester, halide, and anhydride. The activated ester can be selected fromthe group consisting of pentafluorophenol (Pfp), N-hydroxysuccinimide(NHS), N-Hydroxysulfosuccinimide Sodium Salt (NHSS),2-Thioxothiazolidin-1yl, and hydroxybenzotriazole (OBT). The halide canbe selected from the group consisting of F, Cl, Br, and I. The chemicalprotecting group can be selected from the group consisting of Boc, Fmoc,CBZ, t-butyl, benzyl, and allyl.

Converting the precursor MR imaging agent to the MR imaging agent caninclude reacting the precursor imaging agent with a precursor chelatemoiety to form a covalent bond between the precursor chelate moiety andthe linker moiety of the precursor MR imaging agent, the precursorchelate moiety comprising a plurality of carboxylate precursor groups,the carboxylate precursor groups capable of being transformed intocarboxylate moieties; transforming a plurality of the carboxylateprecursor groups of the bound precursor chelate moiety to a plurality ofcarboxylate moieties, the carboxylate moieties capable of complexing aparamagnetic metal ion; and complexing a paramagnetic metal ion to theplurality of carboxylate moieties to produce the MR imaging agent. Theprecursor chelate moiety can be selected from the group consisting of:

wherein Y is a synthetic moiety capable of forming a covalent bond withthe attached linker moiety, and wherein each X, independently, is an O⁻or an O⁻ precursor so that X, upon conversion to O⁻, is capable offorming a carboxylate moiety with its adjacent carbonyl, and R¹ is anuncharged chemical moiety, an aliphatic, alkyl group, or cycloalkylgroup, or uncharged substituted versions thereof. The synthetic moietycan be selected from the group consisting of a carboxylic acid,activated ester, acid halide, anhydride, alkyl halide, isocyanate, andisothiocyanate, and wherein the O⁻ precursor is selected from the groupconsisting of —OH, —OMe, OEt, OtBu, Obenzyl, and O-allyl. The precursorchelate moiety also can be selected from the group consisting of:

wherein LG is a leaving group selected from the group consisting of —OH,activated ester, halide, and anhydride, and wherein each R,independently, is an O⁻ or an O⁻ precursor selected from the groupconsisting of OH, —O—Me, O-Et, O-tBu, O-benzyl, and O-allyl, so that R,upon conversion to O⁻, is capable of forming a carboxylate moiety withits adjacent carbonyl.

The precursor chelate moiety also can be selected from the groupconsisting of:

wherein n is an integer from 1 to 4; R is selected from the groupconsisting of a negative charge and a negative charge precursor capableof being transformed into a negative charge; and X is a chemical leavinggroup selected from the group consisting of —Cl, —Br, —I, -MsO, -TsO,and -TfO.

The precursor chelate moiety can be selected from the group consistingof:

wherein R is selected from the group consisting of a negative charge anda negative charge precursor capable of being transformed into a negativecharge; and X is a chemical leaving group selected from the groupconsisting of —Cl, —Br, —I, -MsO, -TsO, and -TfO. The negative chargeprecursor is selected from the group consisting of —H, -Me, -Et, -t-Bu,-benzyl, and -allyl.

In some embodiments, the linker moiety can be covalently conjugated to aprecursor chelate moiety, the covalent conjugate comprising a pluralityof carboxylate precursor groups, the carboxylate precursor groupscapable of being transformed into carboxylate moieties. Converting theprecursor MRI imaging agent to the MR imaging agent can includetransforming a plurality of the covalent conjugate's carboxylateprecursor groups into carboxylate moieties, the carboxylate moietiescapable of complexing a paramagnetic metal ion; and complexing aparamagnetic metal ion to the plurality of carboxylate moieties toresult in the MR imaging agent. The paramagnetic metal ion can beselected from the group consisting of: Gd(III), Fe(III), Mn(II and III),Cr(III), Cu(II), Dy(III), Tb(III and IV), Ho(III), Er(III), Pr(III),Eu(II) and Eu(III). Gd(III) is a particularly useful paramagnetic ion.

The covalent conjugate can be selected from the group consisting of

wherein n is an integer from 1 to 4; LG is a leaving group selected fromthe group consisting of —OH, activated ester, halide, and anhydride; andR¹, R², R³, R⁴, and R⁵ are independently selected from the groupconsisting of an acetate moiety, a -Me, -Et, or -t-Bu protected acetatemoiety, an acetamide moiety, and an acetoxy moiety.

The covalent conjugate also can be selected from the group consistingof:

wherein LG is a leaving group selected from the group consisting of —OH,activated ester, halide, and anhydride; and R¹, R², R³, and R⁴ areselected from the group consisting of an acetate moiety, a -Me, -Et, or-t-Bu protected acetate moiety, an acetamide moiety, and an acetoxymoiety.

The covalent conjugate can be selected from the group consisting of:

The covalent conjugate also can be selected from the group consistingof:

wherein:R is a -tBu group, LG is a leaving group selected from the groupconsisting of —OH, activated ester, halide, and anhydride.

Methods of the invention further can include, before covalentlyattaching a linker moiety to the C- and N-terminal amine functionalgroups, reacting a linker-subunit with the N-terminal amine functionalgroup of the peptide to produce a derivatized N-terminal aminefunctional group of the peptide. The linker-subunit can be selected fromthe group consisting of:

wherein Base is selected from the group consisting of adenosine,guanosine, thymine, and cytosine; LG is a leaving group selected fromthe group consisting of OH, activated ester, halide, and anhydride; andR is an aliphatic or aromatic moiety. The linker-subunit also can beselected from the group consisting of:

wherein n is independently an integer from 0 to 3; R is an aliphatic oraromatic group; and LG is a leaving group selected from the groupconsisting of: OH, activated ester, halide, and anhydride.

The linker-subunit also can be selected from the group consisting of:

wherein n is independently 1 or 2; R is an aliphatic or aromatic group;and LG is a leaving group selected from the group consisting of: OH,activated ester, halide, and anhydride.

In another aspect, the invention features a method of making a MRimaging agent. The method includes covalently binding an amino acidresidue to a linker-subunit moiety to form a C-terminal end of apeptide, wherein the linker-subunit moiety is covalently attached to aresin; synthesizing a peptide on the resin from the covalently boundC-terminal end to an N-terminal residue of the peptide, the N-terminalresidue comprising an N-terminal amine functional group; cleaving thepeptide from the resin to produce a peptide having a C-terminal aminefunctional group; covalently attaching a linker moiety to the peptide'sC-terminal amine functional group and N-terminal amine functional groupto form a precursor MR imaging agent; and converting the precursor MRimaging agent to the MR imaging agent. The method further can includebefore cleaving the peptide from the resin, covalently attaching alinker-subunit moiety to the N-terminal amino functional group toproduce a derivatized N-terminal amine functional group. The linkermoiety can be covalently conjugated to a precursor chelate moiety, thecovalent conjugate comprising a plurality of carboxylate precursorgroups, the carboxylate precursor groups capable of being transformedinto carboxylate moieties.

Converting the precursor MR imaging agent to the MR imaging agent caninclude reacting the precursor MR imaging agent with a precursor chelatemoiety to form a covalent bond between the precursor chelate moiety andthe linker moiety of the precursor MR imaging agent, the precursorchelate moiety comprising a plurality of carboxylate precursor groups,the carboxylate precursor groups capable of being transformed intocarboxylate moieties; transforming a plurality of the carboxylateprecursor groups of the bound precursor chelate moiety to a plurality ofcarboxylate moieties, the carboxylate moieties capable of complexing aparamagnetic metal ion; and complexing a paramagnetic metal ion to theplurality of carboxylate moieties to produce the MR imaging agent.

Converting the precursor MRI imaging agent to the MR imaging agent alsocan include transforming a plurality of the covalent conjugate'scarboxylate precursor groups into carboxylate moieties, the carboxylatemoieties capable of complexing a paramagnetic metal ion; and complexinga paramagnetic metal ion to the plurality of carboxylate moieties toresult in the MR imaging agent. The paramagnetic metal ion can beselected from the group consisting of: Gd(III), Fe(III), Mn(II and III),Cr(III), Cu(II), Dy(III), Tb(III and IV), Ho(III), Er(III), Pr(III),Eu(II) and Eu(III). Gd(III) is a particularly useful paramagnetic metalion.

In another aspect, the invention features a method of making a MRimaging agent that includes reacting a peptide having a C-terminalcarboxylate functional group with a linker-subunit moiety to form amodified peptide having both a C-terminal carboxylate functional groupand an N-terminal carboxylate functional group; covalently attaching alinker moiety to both the N-terminal and C-terminal carboxylatefunctional groups of the modified peptide to form a precursor MR imagingagent; and converting the precursor MR imaging agent to the MR imagingagent. The linker-subunit moiety can be selected from the groupconsisting of:

wherein LG is a leaving group selected from the group consisting of OH,activated ester, halide, and anhydride; and R is an aromatic oraliphatic group. The linker moiety also can be selected from the groupconsisting of:

wherein m is an integer from 1 to 4; n is an integer from 0 to 4; R isindependently selected from the group consisting of —H, -Me, -Et, -Bz,and -tBu; and R¹ and R² are independently selected from a hydrogen or achemical protecting group.

The linker moiety can be selected from the group consisting of:

wherein R¹ and R² are selected independently from the group consistingof hydrogen and a chemical protecting group, the chemical protectinggroup selected from the group consisting of: Boc, Fmoc, CBZ, t-butyl,benzyl, and allyl.

Converting the precursor MR imaging agent to the MR imaging agent alsocan include reacting the precursor MR imaging agent with a precursorchelate moiety in order to form a covalent bond between the linkermoiety of the precursor MR imaging agent and the precursor chelatemoiety, the precursor chelate moiety comprising a plurality ofcarboxylate precursor groups, the carboxylate precursor groups capableof being transformed into carboxylate moieties; transforming a pluralityof the carboxylate precursor groups of the bound precursor chelatemoiety to a plurality of carboxylate moieties, the carboxylate moietiescapable of complexing a paramagnetic metal ion; and complexing aparamagnetic metal ion to the plurality of carboxylate moieties toproduce the MR imaging agent. The linker moiety can be covalentlyconjugated to a precursor chelate moiety, the covalent conjugatecomprising a plurality of carboxylate precursor groups, the carboxylateprecursor groups capable of being transformed into carboxylate moieties.

Converting the precursor MR imaging agent to the MR imaging agent alsocan include transforming a plurality of the covalent conjugate'scarboxylate precursor groups into carboxylate moieties, the carboxylatemoieties capable of complexing a paramagnetic metal ion; and complexinga paramagnetic metal ion to the plurality of carboxylate moieties toproduce the MR imaging agent.

A covalent conjugate can be selected from the group consisting of:

wherein n is an integer from 1 to 4; and R¹, R², R³, R⁴, and R⁵ areindependently selected from the group consisting of an acetate moiety,a-Me, -Et, or -t-Bu protected acetate moiety, an acetamide moiety, andan acetoxy moiety. The covalent conjugate can be:

Converting the precursor MR imaging agent to the MR imaging agent alsocan include reacting the precursor imaging agent with a chelate moiety,wherein the chelate moiety contains a paramagnetic metal ion, to form acovalent bond between the chelate moiety and the linker moiety of theprecursor MR imaging agent to produce the MR imaging agent. Suitableparamagnetic metal ions are described above.

In yet another aspect, the invention features a contrast agent thatincludes a metal chelate complex at a —CO₂R and NHR termini of abiopolymer (e.g., a peptide), wherein R is independently selected fromthe group consisting of hydrogen, alkyl, aliphatic, and a leaving group.The contrast agent can include two metal chelate complexes at the CO₂Rand NHR termini of the biopolymer. The biopolymer can have a specificbinding affinity for fibrin. The peptide can be capable of forming adisulfide bond under non-reducing conditions, and in some embodiments,includes a disulfide bond. A contrast agent can have the formula:

wherein Chelate represents a metal chelate complex; Linker represents alinker moiety; Linker-subunit represents a linker-subunit moiety; m isindependently an integer from 1 to 10; p is independently an integerfrom 0 to 5; s is independently 0 or 1; R¹ is an amino acid side chainor a derivative thereof; and R² is independently a hydrogen or analiphatic group. A contrast agent also can have a structure of any oneof structures 4-55.

In another aspect, the invention features a method for altering thestability of a peptide, the peptide having an N-terminal aminefunctional group. The method includes reacting the peptide with alinker-subunit moiety to form a peptide having a C-terminal aminefunctional group; and covalently attaching a linker moiety to thepeptide's C-terminal amine functional group and N-terminal aminefunctional group to form a modified peptide. The method further caninclude reacting the modified peptide with a capping moiety to form acovalent bond between the capping moiety and the linker moiety of themodified peptide. The method also can include reacting the modifiedpeptide with a precursor chelate moiety to form a covalent bond betweenthe precursor chelate moiety and the linker moiety of the modifiedpeptide, the precursor chelate moiety comprising a plurality ofcarboxylate precursor groups, the carboxylate precursor groups capableof being transformed into carboxylate moieties. After transforming aplurality of the carboxylate precursor groups of the bound precursorchelate moiety to a plurality of carboxylate moieties, the carboxylatemoieties capable of complexing a paramagnetic metal ion; a paramagneticmetal ion can be complexed to the plurality of carboxylate moieties. Themethod further can include assaying the stability of the modifiedpeptide or assaying the stability of the unmodified peptide andcomparing the stability of said modified peptide to the stability of theunmodified peptide. Stability of the modified peptide can be improvedrelative to the stability of the unmodified peptide (e.g., improved10-fold, 20-fold, or 30-fold relative to the stability of the unmodifiedpeptide). Stability can be assayed using a rat liver homogenate assay.

In another aspect, the invention features a modified peptide having thestructure:

wherein Chelate precursor represents a chelate precursor moiety; Linkerrepresents a linker moiety; Linker-subunit represents a linker-subunitmoiety; m is independently an integer from 1 to 10; p is independentlyan integer from 0 to 5;

-   s is independently 0 or 1; R¹ is an amino acid side chain or a    derivative thereof; and-   R² is selected from the group consisting of H and an aliphatic    group.

In yet another aspect, the invention features a modified peptide havingthe structure:

wherein Linker represents a linker moiety; Linker-subunit represents alinker-subunit moiety; p is independently an integer from 0 to 5; s isindependently 0 or 1; R¹ is an amino acid side chain or a derivativethereof; and R² is selected from the group consisting of H and analiphatic group.

Method of making an MR imaging agent also are featured that includereacting a peptide having an N-terminal amine functional group with alinker-subunit moiety to form a modified peptide having an aminefunctional group on both its N-terminus and C-terminus, or reacting apeptide having a C-terminal carboxylate functional group with alinker-subunit moiety to form a modified peptide having a carboxylatefunctional group on both its C-terminus and N-terminus; and convertingthe modified peptide to the MR imaging agent. Converting the modifiedpeptide to the MR imaging agent can include covalently attaching achelate moiety to the modified peptide, wherein the chelate moietycontains a paramagnetic metal ion, to produce the MR imaging agent.Converting the modified peptide to the MR imaging agent also can includecovalently linking a linker moiety to a chelate moiety to form acovalent conjugate, wherein the chelate moiety contains a paramagneticmetal ion; and reacting the covalent conjugate with the modified peptideto form the MR imaging agent. Suitable paramagnetic ions are describedabove.

In another aspect, the invention features a method of making an MRimaging agent that includes covalently binding an amino acid residue toa linker-subunit moiety to form a C-terminal end of a peptide, whereinthe linker-subunit moiety is covalently attached to a resin;synthesizing a peptide on the resin from the covalently bound C-terminalend to an N-terminal residue of the peptide, the N-terminal residuecomprising an N-terminal amine functional group; cleaving the peptidefrom the resin to produce a C-terminal amine functional group of themodified peptide; converting the modified peptide to the MR imagingagent. Converting the modified peptide to the MR imaging agent caninclude covalently attaching a chelate moiety to the modified peptide,wherein the chelate moiety contains a paramagnetic metal ion, to producethe MR imaging agent. Converting the modified peptide to the MR imagingagent also can include covalently linking a linker moiety to a chelatemoiety to form a covalent conjugate, wherein the chelate moiety containsa paramagnetic metal ion; and reacting the covalent conjugate with themodified peptide to form the MR imaging agent. Suitable paramagneticions are described above.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 provides the chemical structures of non-natural amino acids.

FIG. 2 depicts the relaxivities per Gd at 20 MHz, 35° C. in Trisbuffered saline (TBS) or 10 mg/ml fibrin in TBS.

FIG. 3 depicts the accumulation of a contrast agent in the thrombus.

FIG. 4A is an image of a thrombus. FIG. 4B is an image of a thrombuswith black blood.

DETAILED DESCRIPTION Definitions

Commonly used chemical abbreviations that are not explicitly defined inthis disclosure may be found in The American Chemical Society StyleGuide, Second Edition; American Chemical Society, Washington, D.C.(1997), “2001 Guidelines for Authors” J. Org. Chem. 66(1), 24A (2001),“A Short Guide to Abbreviations and Their Use in Peptide Science” J.Peptide. Sci. 5, 465-471 (1999).

For the purposes of this application, the term “chemical protectinggroup” or “protecting group” means any chemical moiety temporarilycovalently bound to a molecule throughout one or more syntheticchemistry steps in a reaction sequence to prevent undesirable reactions.Common protecting group strategies are described in “Protecting Groupsin Organic Synthesis, Third Ed.” by P. Wuts and T. Greene, © 1999 JohnWiley & Sons, Inc.

For the purposes of this application, the term “leaving group” means anychemical moiety that is displaced by a nucleophile in a nucleophilicsubstitution or sequence of addition-elimination reactions. A moleculecomprising a leaving group may be isolated or it may be formed in situas a transient intermediate in a chemical reaction.

For the purposes of this application, the term “aliphatic” describes anyacyclic or cyclic, saturated or unsaturated, branched or unbranchedcarbon compound, excluding aromatic compounds.

The term “alkyl” includes saturated aliphatic groups, includingstraight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, octyl, nonyl, decyl, etc.), branched-chain alkyl groups(isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups(cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkylsubstituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.The term alkyl further includes alkyl groups, which can further includeoxygen, nitrogen, sulfur or phosphorous atoms replacing one or morecarbons of the hydrocarbon backbone. In certain embodiments, a straightchain or branched chain alkyl has 6 or fewer carbon atoms in itsbackbone (e.g., C₁-C₆ for straight chain, C₃-C₆ for branched chain), andmore preferably 4 or fewer. Likewise, preferred cycloalkyls have from3-8 carbon atoms in their ring structure, and more preferably have 5 or6 carbons in the ring structure. The term C₁-C₆ includes alkyl groupscontaining 1 to 6 carbon atoms.

Moreover, the term “alkyl” includes both “unsubstituted alkyls” and“substituted alkyls,” the latter of which refers to alkyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example,alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbanoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety. Cycloalkyls can be further substituted, e.g.,with the substituents described above. An “arylalkyl” moiety is an alkylsubstituted with an aryl (e.g., phenylmethyl (benzyl)). The term “alkyl”also includes the side chains of natural and unnatural amino acids. Theterm “n-alkyl” means a straight chain (i.e., unbranched) unsubstitutedalkyl group.

The term “alkenyl” includes aliphatic groups that may or may not besubstituted, as described above for alkyls, containing at least onedouble bond and at least two carbon atoms. For example, the term“alkenyl” includes straight-chain alkenyl groups (e.g., ethylenyl,propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl,decenyl, etc.), branched-chain alkenyl groups, cycloalkenyl (alicyclic)groups (cyclopropenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl,cyclooctenyl), alkyl or alkenyl substituted cycloalkenyl groups, andcycloalkyl or cycloalkenyl substituted alkenyl groups. The term alkenylfurther includes alkenyl groups that include oxygen, nitrogen, sulfur orphosphorous atoms replacing one or more carbons of the hydrocarbonbackbone. In certain embodiments, a straight chain or branched chainalkenyl group has 6 or fewer carbon atoms in its backbone (e.g., C₂-C₆for straight chain, C₃-C₆ for branched chain). Likewise, cycloalkenylgroups may have from 3-8 carbon atoms in their ring structure, and morepreferably have 5 or 6 carbons in the ring structure. The term C₂-C₆includes alkenyl groups containing 2 to 6 carbon atoms.

Moreover, the term alkenyl includes both “unsubstituted alkenyls” and“substituted alkenyls,” the latter of which refers to alkenyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example, alkylgroups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moiety.

The term “alkynyl” includes unsaturated aliphatic groups analogous inlength and possible substitution to the alkyls described above, butwhich contain at least one triple bond and two carbon atoms. Forexample, the term “alkynyl” includes straight-chain alkynyl groups(e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl,nonynyl, decynyl, etc.), branched-chain alkynyl groups, and cycloalkylor cycloalkenyl substituted alkynyl groups. The term alkynyl furtherincludes alkynyl groups that include oxygen, nitrogen, sulfur orphosphorous atoms replacing one or more carbons of the hydrocarbonbackbone. In certain embodiments, a straight chain or branched chainalkynyl group has 6 or fewer carbon atoms in its backbone (e.g., C₂-C₆for straight chain, C₃-C₆ for branched chain). The term C₂-C₆ includesalkynyl groups containing 2 to 6 carbon atoms.

In general, the term “aryl” includes groups, including 5- and 6-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, phenyl, pyrrole, furan, thiophene,thiazole, isothiaozole, imidazole, triazole, tetrazole, pyrazole,oxazole, isooxazole, pyridine, pyrazine, pyridazine, and pyrimidine, andthe like. Furthermore, the term “aryl” includes multicyclic aryl groups,e.g., tricyclic, bicyclic, such as naphthalene, benzoxazole,benzodioxazole, benzothiazole, benzoimidazole, benzothiophene,methylenedioxyphenyl, quinoline, isoquinoline, napthridine, indole,benzofuran, purine, benzofuran, deazapurine, or indolizine. Those arylgroups having heteroatoms in the ring structure may also be referred toas “aryl heterocycles,” “heterocycles,” “heteroaryls,” or“heteroaromatics.” An aryl group may be substituted at one or more ringpositions with substituents.

For the purposes of this application, “DTPA” refers to a chemicalcompound comprising a substructure composed of diethylenetriamine,wherein the two primary amines are each covalently attached to twoacetyl groups and the secondary amine has one acetyl group covalentlyattached according to the following formula:

wherein X is a heteroatom electron-donating group capable ofcoordinating a metal cation, preferably O⁻, OH, NH₂, OPO₃ ²⁻; or NHR, orOR wherein R is any aliphatic group. When each X group is tert-butoxy(tBu), the structure may be referred to as “DTPE” (“E” forester).

For the purposes of this application, “DOTA” refers to a chemicalcompound comprising a substructure composed of1,4,7,11-tetraazacyclododecane, wherein the amines each have one acetylgroup covalently attached according to the following formula:

wherein X is defined above.

For the purposes of this application, “NOTA” refers to a chemicalcompound comprising a substructure composed of 1,4,7-triazacyclononane,wherein the amines each have one acetyl group covalently attachedaccording to the following formula:

wherein X is defined above.

For the purposes of this application, “DO3A” refers to a chemicalcompound comprising a substructure composed of1,4,7,11-tetraazacyclododecane, wherein three of the four amines eachhave one acetyl group covalently attached and the other amine has asubstituent having neutral charge according to the following formula:

wherein X is defined above and R¹ is an uncharged chemical moiety,preferably hydrogen, any aliphatic, alkyl group, or cycloalkyl group,and uncharged derivatives thereof. The preferred chelate “HP”-DO3A hasR¹═—CH₂(CHOH)CH₃.

In each of the four structures above, the carbon atoms of the indicatedethylenes may be referred to as “backbone” carbons. The designation“bbDTPA” may be used to refer to the location of a chemical bond to aDTPA molecule (“bb” for “back bone”). Note that as used herein,bb(CO)DTPA-Gd means a C═O moiety bound to an ethylene backbone carbonatom of DTPA.

The terms “chelating ligand,” “chelating moiety,” and “chelate moiety”may be used to refer to any polydentate ligand which is capable ofcoordinating a metal ion, including DTPA (and DTPE), DOTA, DO3A, or NOTAmolecule, or any other suitable polydentate chelating ligand as isfurther defined herein, that is either coordinating a metal ion or iscapable of doing so, either directly or after removal of protectinggroups, or is a reagent, with or without suitable protecting groups,that is used in the synthesis of a contrast agent and comprisessubstantially all of the atoms that ultimately will coordinate the metalion of the final metal complex. The term “chelate” refers to the actualmetal-ligand complex, and it is understood that the polydentate ligandwill eventually be coordinated to a medically useful metal ion.

The term “specific binding affinity” as used herein, refers to thecapacity of a contrast agent to be taken up by, retained by, or bound toa particular biological component to a greater degree than othercomponents. Contrast agents that have this property are said to be“targeted” to the “target” component. Contrast agents that lack thisproperty are said to be “non-specific” or “non-targeted” agents. Thespecific binding affinity of a binding group for a target is expressedin terms of the equilibrium dissociation constant “Kd.”

The term “relaxivity” as used herein, refers to the increase in eitherof the MRI quantities 1/T1 or 1/T2 per millimolar (mM) concentration ofparamagnetic ion or contrast agent, which quantities may be different ifthe contrast agent contains a multiplicity of paramagnetic ions, whereinT1 is the longitudinal or spin-lattice, relaxation time, and T2 is thetransverse or spin-spin relaxation time of water protons or otherimaging or spectroscopic nuclei, including protons found in moleculesother than water. Relaxivity is expressed in units of mM⁻¹s⁻¹.

The term “open coordination site” as used herein refers to a site on ametal ion that is generally occupied by a water or solvent molecule.

As used herein, the term “purified” refers to a peptide that has beenseparated from either naturally occurring organic molecules with whichit normally associates or, for a chemically-synthesized peptide,separated from any other organic molecules present in the chemicalsynthesis. Typically, the polypeptide is considered “purified” when itis at least 70% (e.g., 70%, 80%, 90%, 95%, or 99%), by dry weight, freefrom any other proteins or organic molecules.

As used herein, the term “peptide” refers to a chain of amino acids thatis about 2 to about 75 amino acids in length (e.g., 3 to 50 aminoacids).

As used herein, the term “biopolymer” refers to a polymeric substancethat is naturally formed in a biological system. Certain biopolymers canbe constructed from a defined set of building subunits and with commonfunctionalities linking the subunits, e.g., a peptide is usuallyconstructed from a set of amino acids (both natural and non-natural)with amide bonds linking the subunits.

The term “multimer” for purposes herein is defined as a contrast agentor a subunit thereof comprising at least two covalently bonded chelatesor synthetic precursors thereof.

As used herein, the term “natural” or “naturally occurring” amino acidrefers to one of the twenty most common occurring amino acids. Naturalamino acids modified to provide a label for detection purposes (e.g.,radioactive labels, optical labels, or dyes) are considered to benatural amino acids. Natural amino acids are referred to by theirstandard one- or three-letter abbreviations.

The term “non-natural amino acid” or “non-natural” refers to anyderivative of a natural amino acid including D forms, and β and γ aminoacid derivatives. It is noted that certain amino acids, e.g.,hydroxyproline, that are classified as a non-natural amino acid herein,may be found in nature within a certain organism or a particularprotein.

The term “stable,” as used herein, refers to compounds that possessstability sufficient to allow manufacture and which maintains theintegrity of the compound for a sufficient period of time to be usefuland safe for the purposes detailed herein. Typically, such compounds arestable at a temperature of 40° C. or less, in the absence of moisture orother chemically reactive conditions, for at least a week. Combinationsof substituents and variables envisioned by this invention are onlythose that result in the formation of stable compounds.

The terms “target binding” and “binding” for purposes herein refer tonon-covalent interactions of a contrast agent with a target. Thesenon-covalent interactions are independent from one another and may be,inter alia, hydrophobic, hydrophilic, dipole-dipole, pi-stacking,hydrogen bonding, electrostatic associations, or Lewis acid-baseinteractions.

The term “capping moiety” refers to a chelate, organic dye, contrastagent, thrombolytic, or stabilizing moiety. Suitable stabilizingmoieties are biologically inert, i.e., does not have biologicalactivity.

Contrast Agents

In general, the present invention relates to MRI, optical, andradionuclide contrast agents that include a targeting polymer (e.g.,peptide) in which both the N- and C-terminal amino acids are eachconjugated, either directly or via an optional interveninglinker-subunit and linker, to at least one chelate of a paramagnetic(for magnetic resonance imaging) or radioactive (for radionuclideimaging) metal ion or an optical dye (for optical imaging). As furtherexemplified herein, the linker or linker-subunit may be branched andtherefore allow for multiple chelates or dyes to be attached to each endof the peptide, i.e. a multimer. The compounds of this invention maycontain one or more asymmetric carbon atoms and thus may occur asracemates and racemic mixtures, single enantiomers, diastereomericmixtures and individual diastereomers. All such isomeric forms of thesecompounds are expressly included in the present invention. Eachstereogenic carbon may be of the R or S configuration unlessspecifically designated otherwise. Although the specific compoundsexemplified in this application may be depicted in a particularstereochemical configuration, compounds having either the oppositestereochemistry at any given chiral center or mixtures thereof are alsoenvisioned. It should be understood that the compounds of this inventionmay adopt a variety of conformational and ionic forms in solution, inpharmaceutical compositions and in vivo. Although the depictions hereinof specific preferred compounds of this invention are of particularconformations and ionic forms, the disclosure of the invention is not solimited.

Novel peptide-based multimers of the present invention offer severaladvantages as targeted contrast agents.

-   -   1. The compounds can deliver two or more capping moieties (e.g.,        chelates, organic dyes, or thrombolytics) to the target using a        single targeting peptide so that sufficient improvement in the        tissue contrast will be observed in part because of a meaningful        concentration of the imaging moiety around the target.    -   2. The MRI contrast agents of this invention also exhibit a high        relaxivity upon binding to the target due to the receptor        induced magnetic enhancement (RIME) effect combined with the        ability of the peptide to limit the local motion of individual        chelates when bound to the target.    -   3. The compounds have a high affinity for one or more targets.    -   4. Since the compounds are relatively easy to synthesize        according to the methods described herein and only one peptide        per molecule is required, a multiplicity of metal ions or        organic dyes may be delivered to a target more economically.    -   5. The compounds of the invention can have higher in vivo        stability (i.e., longer half-lives) from diminished enzyme        metabolism (e.g., decreased cleavage by peptidases).

These favorable features of peptide-based multimers according to thepresent invention make them useful targeted contrast agents.

The chemical structure of MRI and radionuclide contrast agentscontemplated by the invention may be illustrated by the formula:

wherein for each m, independently, 1≦m≦10, chelate represents a metalchelate complex, p is independently an integer from zero to five; s isindependently one or zero; R¹ is any amino acid side chain includingside chains of non-natural amino acids; R² is any aliphatic group orhydrogen; and n is an integer from 3 to 50 inclusive. Alternately, R¹and R² may be taken together to form a ring structure (including prolineand substituted versions thereof). Linkers, if present, may bedifferent.

Metal ions preferred for MRI include those with atomic numbers 21-29,39-47, or 57-83, and, more preferably, a paramagnetic form of a metalion with atomic numbers 21-29, 42, 44, or 57-83. Particularly preferredparamagnetic metal ions are selected from the group consisting ofGd(III), Fe(III), Mn(II and III), Cr(III), Cu(II), Dy(II), Tb(III andIV), Ho(III), Er(III), Pr(III) and Eu(II and III). Gd(II) isparticularly useful. Note that, as used herein, the term “Gd” is meantto convey the ionic form of the metal gadolinium; such an ionic form canbe written as GD(III), GD3+, gado, etc., with no difference in ionicform contemplated.

For radionuclide imaging agents, radionuclides ⁹⁰Y, ^(99m)Tc, ¹¹¹In,⁴⁷Sc, ⁶⁷Ga, ⁵¹Cr, ^(177m)Sn, ⁶⁷Cu, ¹⁶⁷Tm, 97Ru, ¹⁸⁸Re, ¹⁷⁷Lu, 199Au,²⁰³Pb, and ¹⁴¹Ce are particularly useful. Metal complexes with usefuloptical properties also have been described. See, Murru et al., J. Chem.Soc. Chem. Comm. 1993, 1116-1118. For optical imaging using chelates,lanthanide chelates such as La(III), Ce(III), Pr(III), Nd(III), Pn(III),Sm(III), Eu(III), Gd(III), Tb(III), Dy(III), Ho(III), Er(III), Tm(II),Yb(III) and Ln(III) are suitable. Eu(III) and Tb(III) are particularlyuseful.

Metal chelates should not dissociate to any significant degree duringthe imaging agent's passage through the body, including while bound to atarget tissue. Significant release of free metal ions can result intoxicity, which would generally not be acceptable.

In one embodiment, with reference to the above structure of a contrastagent, m is 2, n, s, R¹, and R² are defined as above, and the Linkermoiety comprises:

The “Chelate” is preferably bb(CO)DTPA.Gd.

In another embodiment, with reference to the above structure of acontrast agent, m is 2, n, s, R¹, and R² are defined as above, and thelinker moiety comprises:

The “Chelate” moiety can be bb(CO)DTPA.Gd.

For the purposes of illustration, one contrast agent contemplated by theinstant invention is presented below with the various subunitsannotated:

wherein R=amino acid side chains such that the peptide has affinity fora biological target, and m=metal ion (paramagnetic for MRI, radioactivefor radionuclide imaging, and fluorescent, luminescent, or absorbant foroptical imaging).

The chemical structure of optical contrast agents contemplated by theinvention may be illustrated by the formula:

wherein 1≦m≦10, p is independently an integer from zero to five, n is 3to 50 inclusive, R¹ is any amino acid side chain including non-naturalamino acid side chains, and R² is any aliphatic group or hydrogen.Alternatively, R¹ and R² taken together form a ring structure (includingPro and derivatives thereof. The N- and C-terminal amino acids of thepeptide can be conjugated to the optical dye directly or via an optionallinker (e.g., p=0 or 1). The linker moieties can be different.

The optical dye may be an organic dye or an appropriate metal chelate.Organic dyes suitable for optical imaging have been described andinclude, for example, fluorescent porphyrin and fluorescentphthalocyanines [see, e.g., U.S. Pat. No. 5,641,878], particulatematerials [see, e.g., WO 96/23524], and polymethine dyes [see, e.g., WO97/13490]. Commonly used optical organic dyes are fluorescein, rhodamine[see, e.g., Kojima H., et al., Anal. Chem. 73, 1967-1973 (2001)],tetramethylrhodamine [e.g., Anal. Biochem. 223, 39 (1994)], and Texasred [e.g., Proc. Natl. Acad. Sci. USA 85, 3546 (1988)]. Fluoroscein andluminescent lanthanide chelates are particularly useful.

Targets and Target Binding Peptides

The peptide moiety of the contrast agents of the present invention canexhibit specific binding for a biological target and function as a pointof attachment for one or more chelates at each terminus. In general,biological targets are present in a low (e.g., micromolar or less)concentration and are inefficiently imaged using existing monomericgadolinium complex MRI contrast agents. The peptide-based multimeric MRIcontrast agents according to the instant invention, however, provide amuch higher concentration of the agent at the target as well as is highrelaxivity to make imaging of these targets possible. Similarly, thepeptide-based multimeric radionuclide contrast agents of the inventionmay deliver more radionuclides to targets so that imaging can be furtherimproved. While not being bound to a particular mechanism, it is thoughtthat targeting creates an increased concentration of the imaging agentat the site to be imaged and increases the relaxivity of MRI contrastagents in the bound state through the RIME effect and also limits localchelate motion by rigidifying the bound peptide.

Targets for the contrast agents can be in any body compartment, cell,organ, or tissue or component thereof. Preferred targets are those thatare of diagnostic and therapeutic relevance, i.e., those that areassociated with disease states. Particularly preferred targets are thosein association with body fluids, and particularly those in associationwith blood, plasma, lymph and fluids of the central nervous system.Other preferred targets are proteins and receptors that either exist inhigh concentration or have a large number of binding sites for certainligands. Included among such target proteins are enzymes andglycoproteins.

Human serum albumin (HSA) and fibrin are useful targets for MRI contrastagents. For vascular blood pool imaging, serum albumin is a preferredtarget. Since HSA is present at high concentration in serum(approximately 0.6 mM) and binds a wide array of molecules withreasonably high affinity, it is a preferred target plasma protein forblood pool contrast agents. HSA is a particularly preferred target forcardiovascular imaging; see U.S. patent application Ser. No. 08/875,365,filed Jul. 24, 1997, and WO 96/23526.

For imaging thrombi, fibrin is a preferred target because it is presentin all clots and it can be targeted without interfering with the normalthrombolytic process. For additional details concerning target bindingmoieties that include fibrin-binding peptides, see PCT PatentApplication WO 01/09188.

Other protein targets include, but are not limited to, alpha acidglycoprotein, fibrinogen, collagen, platelet GPIIb/IIIa receptor,chemotactic peptide receptor, somatostatin receptors, vasoactiveintestinal peptides (VIP) receptor, bombesin/Gastrin release peptidereceptor, and integrin receptors.

Suitable peptides for use in the invention include those capable ofspecifically binding to the targets identified above. Included amongsuch peptides are RGD-containing peptides targeting platelet GPIIb/IIIareceptor for thrombus imaging, chemotactic peptides targeting whiteblood cells for infection/inflammation imaging, Octreotide and P-829peptide targeting somastatin receptors for tumor imaging, vasoactiveintestinal peptides (VIP) targeting VIP receptor for tumor imaging,bombesin analogs targeting bombesin/Gastrin release peptide receptor fortumor imaging, and RGD-containing peptides targeting the integrin αvβ3(vitronectin receptor) for tumor imaging.

In principle, any peptide with an affinity for a biological target maybe used in a contrast agent of the invention. The peptide may be linearor cyclic. Ordinarily, insoluble lipophilic peptides are consideredunsuitable for pharmacological use, but such peptides may be suitableaccording to the invention because addition of hydrophilic metalchelates to the two termini of the peptide may increase solubility. Forease of synthesis and cost considerations, it is preferred that thepeptides have between 3 to 50 amino acids (e.g., 3 to 30, 3 to 20, 3 to15, 5 to 30, 5 to 20, 5 to 15, 10 to 12 amino acids in length).

In the targeting peptides of the invention, a great variety of aminoacids can be used. Suitable amino acids include natural and non-naturalamino acids. Amino acids with many different protecting groupsappropriate for immediate use in the solid phase synthesis of peptidesare commercially available. In addition to the twenty most commonnaturally occurring amino acids, the following non-natural amino acidsor amino acid derivatives may be constituents of the peptide targetinggroup of the invention (common abbreviations in parentheses, see FIG.1): β-Alanine (β-Ala), γ-Aminobutyric Acid (GABA), 2-Aminobutyric Acid(2-Abu), α,β-Dehydro-2-aminobutyric Acid (Δ-Abu),1-Aminocyclopropane-1-carboxylic Acid (ACPC), Aminoisobutyric Acid(Aib), 2-Amino-thiazoline-4-carboxylic Acid, 5-Aminovaleric Acid(5-Ava), 6-Aminohexanoic Acid (6-Ahx), 8-Aminooctanoic Acid (8-Aoc),11-Aminoundecanoic Acid (11-Aun), 12-Aminododecanoic Acid (12-Ado),2-Aminobenzoic Acid (2-Abz), 3-Aminobenzoic Acid (3-Abz), 4-AminobenzoicAcid (4-Abz), 4-Amino-3-hydroxy-6-methylheptanoic Acid (Statine, Sta),Aminooxyacetic Acid (Aoa), 2-Aminotetraline-2-carboxylic Acid (Atc),4-Amino-5-cyclohexyl-3-hydroxypentanoic Acid (ACHPA),para-Aminophenylalanine (4-NH2-Phe), Biphenylalanine (Bip),para-Bromophenylalanine (4-Br-Phe), ortho-Chlorophenylalanine(2-Cl-Phe), meta-Chlorophenylalanine (3-Cl-Phe),para-Chlorophenylalanine (4-Cl-Phe), meta-Chlorotyrosine (3-Cl-Tyr),para-Benzoylphenylalanine (Bpa), tert-Butylglycine (Tle),Cyclohexylalanine (Cha), Cyclohexylglycine (Chg), 2,3-DiaminopropionicAcid (Dpr), 2,4-Diaminobutyric Acid (Dbu), 3,4-Dichlorophenylalanine(3,4-C12-Phe), 3,4-Diflurorphenylalanine (3,4-F2-Phe),3,5-Diiodotyrosine (3,5-I2-Tyr), ortho-Fluorophenylalanine (2-F-Phe),meta-Fluorophenylalanine (3-F-Phe), para-Fluorophenylalanine (4-F-Phe),meta-fluorotyrosine (3-F-Tyr), Homoserine (Hse), Homophenylalanine(Hfe), Homotyrosine (Htyr), 5-Hydroxytryptophan (5-OH-Trp),Hydroxyproline (Hyp), para-Iodophenylalanine (4-I-Phe), 3-lodotyrosine(3-I-Tyr), Indoline-2-carboxylic Acid (Idc), Isonipecotic Acid (Inp),meta-methyltyrosine (3-Me-Tyr), I-Naphthylalanine (1-Nal), 2Naphthylalanine (2-Nal), para-Nitrophenylalanine (4-NO2-Phe),3-Nitrotyrosine (3-NO2-Tyr), Norleucine (Nle), Norvaline (Nva), Omithine(Orn), ortho-Phosphotyrosine (H2PO3-Tyr), Octahydroindole-2-carboxylicAcid (Oic), Penicillamine (Pen), Pentafluorophenylalanine (F5-Phe),Phenylglycine (Phg), Pipecolic Acid (Pip), Propargylglycine (Pra),Pyroglutamic Acid (pGlu), Sarcosine (Sar),Tetrahydroisoquinoline-3-carboxylic Acid (Tic), andThiazolidine-4-carboxylic Acid (Thioproline, Th). Stereochemistry ofamino acids may be designated by preceding the name or abbreviation withthe designation “D” or “d” or “L” or “l” as appropriate. Additionally,αN-alkylated amino acids may be employed, as well as amino acids havingamine-containing side chains (such as Lys and Orn) in which the aminehas been acylated or alkylated.

Peptides of the invention can include the general formula P*—Y*—X₁*-L*(SEQ ID NO:1), wherein P* is a proline or a non-natural derivative ofproline, Y* is a tyrosine or a non-natural derivative thereof, X₁* isglycine or aspartic acid, or a non-natural derivative of glycine oraspartic acid, and L* is leucine or a non-natural derivative thereof.Typically, at least one of P*, Y*, X₁*, or L* is a non-naturalderivative of the respective amino acid. For example, X₁* can be glycineor aspartic acid, L* can be leucine, and at least one of P* or Y* can bea non-natural derivative, such as hydroxyproline or a tyrosinesubstituted at the 3 position with F, Cl, Br, I, or NO₂.

A peptide of the invention also can include the general formulaX₁—X₂—C—P*—Y*—X₃-L-C—X₄—X₅—X₆ (SEQ ID NO:2), wherein P* is a proline ora non-natural derivative thereof; Y* is a tyrosine or a non-naturalderivative thereof; X₁ is W, Y, F, S, Bip, Hx, Dpr, Cy, Gu, Ad, Hfe,3-Pal, 4-Pal, DopaMe2, nTyr, dW, dF, F(3/4*), orY(3*). F(3/4*) can be aphenylalanine substituted at either the 3 or the 4 position with amoiety such as CH₃, CF₃, NH₂, CH₂NH₂, CN, F, Cl, Br, I, Et, or Ome.Y(3*) can be a tyrosine substituted at the 3 position with a moiety suchas F, Cl, Br, I, and NO₂. X₂ can be E, H, dE, S, H(Bzl), 2-Pal, Dpr, orTh; X₃ can be G or D; X₄ can be H, F, Y, or W; X₅ can be I, L, V, N,Bpa, Bal, Hfe, Nle, Tle, Nval, Phg, Cha, Taz, Fua, Th, 4-Pal, orF(3/4*), wherein F(3/4*) is a phenylalanine substituted at either the 3or the 4 position with a moiety such as CF₃, Et, iPr, or OMe; X₆ can beN, Q, I, L, or V, or not present. Typically, at least one of X₁, X₂, X₅,P*, and Y* is a non-natural derivative of an amino acid. For example, P*can be proline and Y* can be a non-natural derivative of tyrosinesubstituted at the 3 position with a moiety such as F, Cl, Br, I, orNO₂. Alternatively, P* can be a non-natural derivative of proline suchas 4-hydroxyproline and Y* can be tyrosine. Such peptides can form adisulfide bond under non-reducing conditions.

Another example of a peptide that can bind fibrin includes the generalformula C—P*—Y*—X₁-L-C (SEQ ID NO:3), wherein X₁ is G or D, P* isproline or its non-natural derivative 4-hydroxyproline; Y* is tyrosineor a non-natural derivative of tyrosine substituted at the 3 positionwith a moiety such as F, Cl, Br, I, or NO₂. Typically, at least one ofP* or Y* is a non-natural derivative of the respective amino acid. Forexample, the peptide can have the following sequences:W-dE-C—P(4-OH)—Y(3-Cl)-G-L-C—W—I-Q (SEQ ID NO:4),Y-dE-C—P(4-OH)—Y(3-Cl)-G-L-C—Y—I-Q (SEQ ID NO:5),Y-dE-C—P(4-OH)—Y(3-Cl)-G-L-C—W—I-Q (SEQ ID NO:6),W-dE-C—P(4-OH)—Y(3-Cl)-G-L-C—Y—I-Q (SEQ ID NO:7),W-dE-C—P(4-OH)—Y(3-Cl)-D-L-C—W—I-Q (SEQ ID NO:8),Y-dE-C—P(4-OH)—Y(3-Cl)-D-L-C—Y—I-Q (SEQ ID NO:9),Y-dE-C—P(4-OH)—Y(3-Cl)-D-L-C—W—I-Q (SEQ ID NO:10),W-dE-C—P(4-OH)—Y(3-Cl)-D-L-C—Y—I-Q (SEQ ID NO:11),F(4-OMe)—H—C—P(4-OH)—Y(3-Cl)-D-L-C—H—I-L (SEQ ID NO:12),Y—H—C—P(4-OH)—Y(3-Cl)-G-L-C—W—I-Q (SEQ ID NO:13),W-dE-C—P—Y(3-Cl)-G-L-C—W—I-Q (SEQ ID NO:14),W-dE-C—P(4-OH)—Y-G-L-C—W—I-Q (SEQ ID NO:15), orF—H—C—P-(4-OH)—Y(3-Cl)-D-L-C—H—I-L (SEQ ID NO:16). Such peptides canform disulfide bonds under non-reducing conditions.

According to standard synthesis methods such as those disclosed in WO01/09188 or in WO 01/08712, peptides having the sequence set forth inTable 1 were synthesized (structure confirmed by mass spectrometry),cyclized, and assayed for affinity to the DD(E) fragment of fibrin. Eachpeptide was found to have a Kd≦10 μM (“-” indicates truncation).

TABLE 1 Kd(μM) vs. DD(E) X₁ X₂ C P(4-OH) Y* X₃ L C X₄ X₅ X₆ ≦0.1F(4-OMe) H C Hyp Y(3-Cl) D L C H I L ≦0.1 F(4-OMe) H C Hyp Y(3-Cl) D L CH I ≦0.1 F(4-OMe) H C Hyp Y(3-I) D L C H Bpa ≦0.1 F H C Hyp Y(3-I) D L CH Hfe ≦0.1 F H C Hyp Y(3-I) D L C H Bpa ≦0.1 Y(3-Cl) H C Hyp Y(3-I) D LC H I ≦0.1 Y D-E C Hyp Y(3-Cl) G L C W I Q ≦0.1 F(4-OMe) H C Hyp Y(3-I)D L C H I L ≦0.1 F(4-OMe) H(Bzl) C Hyp Y(3-Cl) D L C H Bpa ≦0.1 F H CHyp Y(3-Cl) D L C H I ≦0.1 F(4-OMe) H C Hyp Y(3-I) D L C H I ≦0.1 3Pal HC Hyp Y(3-I) D L C H I ≦0.1 4Pal H C Hyp Y(3-I) D L C H I ≦0.1 F(4-F) HC Hyp Y(3-I) D L C H I ≦0.1 Y(3-I) H C Hyp Y(3-I) D L C H I ≦0.1 F H CHyp Y(3-I) D L C H I L ≦0.1 F(4-OMe) H C Hyp Y(3-Cl) D L C H Bpa L ≦0.1F(4-OMe) H C Hyp Y(3-I) D L C H Bpa L ≦0.1 F H(Bzl) C Hyp Y(3-Cl) D L CH I L ≦0.1 1Nal H C Hyp Y(3-I) D L C H I ≦0.1 MTyr H C Hyp Y(3-I) D L CH I ≦0.1 F(4-OMe) H(Bzl) C Hyp Y(3-I) D L C H Bpa ≦0.1 F(4-OMe) H(Bzl) CHyp Y(3-Cl) D L C H I ≦0.1 F H C Hyp Y(3-I) D L C 3Pal I ≦0.1 F(4-I) H CHyp Y(3-I) D L C H I ≦0.1 F(4-Br) H C Hyp Y(3-I) D L C H I ≦0.1 F(4-Me)H C Hyp Y(3-I) D L C H I ≦0.1 F(4-CF3) H C Hyp Y(3-I) D L C H I ≦0.1F(4-CN) H C Hyp Y(3-I) D L C H I ≦0.1 Y(3-NO2) H C Hyp Y(3-I) D L C H I≦0.1 Y(2-F) H C Hyp Y(3-I) D L C H I ≦0.1 F(4-CH2NH2) H C Hyp Y(3-I) D LC H I ≦0.1 F(4-NH2) H C Hyp Y(3-I) D L C H I ≦0.1 F(34-F2) H C HypY(3-I) D L C H I ≦0.1 DopaMe2 H C Hyp Y(3-I) D L C H I ≦0.1 F(2-OMe) H CHyp Y(3-I) D L C H I ≦0.1 F(3-Me) H C Hyp Y(3-I) D L C H I ≦0.1 F H CHyp Y(3-I) D L C H I ≦0.1 F H C Hyp Y(3-I) D L C H F(3-CF3) ≦0.1F(3-CF3) H C Hyp Y(3-I) D L C H I ≦0.1 F(3-OMe) H C Hyp Y(3-I) D L C H I≦0.1 Hfe H C Hyp Y(3-I) D L C H I ≦0.1 nTyr H C Hyp Y(3-I) D L C H I≦0.1 W E C Hyp Y(3-Cl) G L C W I Q ≦0.1 F H C Hyp Y(3-I) D L C H I L≦0.1 F H C Hyp Y(3-Cl) D L C H I L ≦0.1 F(4-OMe) H(Bzl) C Hyp Y(3-I) D LC H I ≦0.1 F H C Hyp Y(3-I) D L C H Nle ≦0.1 F H C Hyp Y(3-I) D L C HTle ≦0.1 F H C Hyp Y(3-I) D L C H F(4-CF3) ≦0.1 F H C Hyp Y(3-I) D L C HBip ≦0.1 F H C Hyp Y(3-I) D L C H F(4-Et) ≦0.1 F H C Hyp Y(3-I) D L C HF(4-OMe) ≦0.1 F H C Hyp Y(3-I) D L C H F(3-OMe) ≦0.1 F(F5) H C HypY(3-I) D L C H I ≦0.1 F H C Hyp Y(3-F) D L C H I L ≦0.1 W E C HypY(3-Cl) G L C W I Q ≦0.2 T D-E C Hyp Y(3-Cl) G L C W I Q ≦0.2 F H C PY(3-Cl) D L C H I L ≦0.2 Y(26-Me) H C Hyp Y(3-I) D L C H I ≦0.2 W E CHyp Y(3-Cl) G L C H I Q ≦0.2 D-F D-E C Hyp Y(3-Cl) G L C W I Q ≦0.2 Y EC Hyp Y(3-Cl) G L C Y I Q ≦0.2 W E C Hyp Y(3-Cl) G L C F I Q ≦0.2 H D-EC Hyp Y(3-Cl) G L C W I Q ≦0.2 F H C Hyp Y(3-I) D L C H I L ≦0.2 W E C PY G L C W I Q ≦0.2 F H C Hyp Y(3-I) D L C H nVal ≦0.2 F H C Hyp Y(3-I) DL C H Phg ≦0.2 F H C Hyp Y(3-I) D L C H F(3-Me) ≦0.2 F H C Hyp Y(3-I) DL C 4Pal I ≦0.2 S D-E C Hyp Y(3-Cl) G L C W I Q ≦0.3 W E C Hyp Y(3-Cl) GL C Y I Q ≦0.3 Y E C Hyp Y(3-Cl) G L C W I Q ≦0.3 F D-E C Hyp Y(3-Cl) GL C W I Q ≦0.3 F H C P Y D L C H I L ≦0.3 F H C Hyp Y(3-I) D L C H I L≦0.3 F H C Hyp Y D L C H Bpa ≦0.4 F H C Hyp Y(3-Cl) G L C H I L ≦0.4S(Bzl) H C P Y D L C H I L ≦0.4 H E C Hyp Y(3-Cl) G L C H I Q ≦0.4F(4-OMe) H C Hyp Y D L C H I L ≦0.4 F H C Hyp Y(3-I) D L C Bpa I ≦0.4 AdH C Hyp Y(3-I) D L C H I ≦0.5 F H C Hyp Y(3-Cl) F L C H I L ≦0.5 F E CHyp Y(3-Cl) G L C W I Q ≦0.5 F H C Hyp Y(3-Cl) 2-Nal L C H I L ≦0.5 F HC Hyp Y D L C H I L ≦0.5 Hfe H C Hyp Y D L C H I L ≦0.5 Bip H C Hyp Y DL C H I L ≦0.5 W E C P Y G L C W I Q ≦0.5 F(4-OMe) W C Hyp Y(3-I) D L CH I ≦0.5 F H C Hyp Y(3-I) D L C 2Pal I ≦0.5 F H C Hyp Y(3-I) D L C Taz I≦0.5 F H C Hyp Y(3-I) D L C Dht I ≦0.5 Gu H C Hyp Y(3-I) D L C H I

A peptide also can have the general formula C-D-Y—Y-G-T-C—X₁₀ (SEQ ID.NO:17), wherein X₁₀ is n(decyl)G, n(4-PhBu)G, MeL, Bpa, Bip, Me-Bip,F(4*), F(3-Me), F(3,4-difluoro), Amh, Hfe, Y(3,5-di-iodo), Pff, 1Nal,d1Nal, or MeL, wherein F(4*) is a phenylalanine substituted at the 4position with a moiety such as Et, CF₃, I, or iPr. In some embodiments,a peptide can include additional residues, X₁, P*, and/or X₁₁, toprovide the general formula: C-D-Y—Y-G-T-C—X₁₀—X₁ (SEQ ID. NO:18) orX—P*—C-D-Y—Y-G-T-C—X₁₀—X₁₁ (SEQ ID. NO:26), wherein X₁ is any natural ornon-natural amino acid, P* is proline or a non-natural derivativethereof, and X₁₁ is D, dD, βD, Inp, Nip, Me-D, Cop, or Cmp. For example,a peptide can have the sequence of L-P—C-D-Y—Y-G-T-C-n(Decyl)G-dD (SEQID NO:19), L-P—C-D-Y—Y-G-T-C-n(Decyl)G-D (SEQ ID NO:20),L-P—C-D-Y—Y-G-T-C-Bip-D (SEQ ID NO:21), L-P—C-D-Y—Y-G-T-C-Bip-dD (SEQ IDNO:22), L-P—C-D-Y—Y-G-T-C-MeL-Inp (SEQ ID NO:23),L-P—C-D-Y—Y-G-T-C-MeL-Cmp (SEQ ID NO:24), or L-P—C-D-Y—Y-G-T-C-MeBip-D(SEQ ID NO:25).

Peptides having the formula of SEQ ID NO:26 were synthesized (structureconfirmed by mass spectrometry) according to standard synthesis methods,such as those disclosed in WO 01/09188 or in WO 01/08712, and assayedfor affinity to the DD(E) fragment of fibrin. Each peptide was found tohave a Kd≦10 μM (Table 2).

TABLE 2 X₀₁ X₁₀ X₁₁ L n(Decyl)G dD L n(Decyl)G D L MeL Inp L Bip D L BipdD L Me-Bip D L MeL Cmp L Bip D L L D Cha Bip D

The ability of the peptides to bind a target such as HSA or fibrin canbe assessed by known methodology. For example, affinity of the peptidefor fibrin can be assessed using the DD(E) fragment of fibrin, whichcontains subunits of 55 kD (Fragment E) and 190 kD (Fragment DD). TheDD(E) fragment can be biotinylated and immobilized via avidin to a solidsubstrate (e.g., a multi-well plate). Peptides can be incubated with theimmobilized DD(E) fragment in a suitable buffer and binding detectedusing known methodology. See, for example, WO 01/09188.

N- and C-Terminus Linker-Subunits and Linker

If present, linker-subunits and linkers are used to covalently attachcapping moieties such as chelates, thrombolytics, and other groups tothe two ends of a peptide. A linker-subunit moiety can (i) convert thefunctionality of either the C-terminus carboxylate to an aminefunctional group or the N-terminus amine to a carboxylate functionalgroup; or (ii) provide a spacer moiety or group between the peptideterminus and the linker, if present, or capping group. In oneembodiment, a peptide can be reacted with a linker-subunit to form amodified peptide having a C-terminal amine functional group and aN-terminal amine functional group. In another embodiment, a peptide canbe reacted with a linker-subunit to form a modified peptide having aN-terminal carboxylate functional group and a C-terminal carboxylatefunctional group. In another embodiment, a peptide can be synthesizedfrom a C-terminal linker-subunit that is bound to a resin, whereby uponcleaving the peptide from the resin, a peptide having a C-terminal aminefunctional group is produced. In still another embodiment, alinker-subunit can be used as a spacer group and not to change theterminus functional group. A linker-subunit may have multiple functionalgroups for attachment of linker moieties or capping moieties. Many typesof reactions can be used, including acylation, reductive anination,nucleophilic displacement reactions, urea formation, thiourea formation,and chemoselective ligation in chemically conjugating the linker-subunitto the peptide, linker, and/or capping moieties. One advantage of usinga linker-subunit is to create similar functional groups on the peptide,thereby facilitating subsequent synthesis.

The linker moiety can be used to covalently attach one or more cappingmoieties to the peptide terminus. The linker may be branched orunbranched and may comprise multiple functional groups for precursorchelate and chelate attachment. The chemical structure of the linker mayaffect the physical and pharmacological properties of the contrastagent, such as affinity, stability, blood half-life, relaxivity, andplasma protein binding. Linkers may be substituted with alkyl, aryl,alkenyl, or alkynyl groups. Linkers, if present, at each termini,typically are relatively small and rigid for MRI contrast agents. Forexample, a linker can have a molecular weight less than about 350 (e.g.,less than about 200).

An example of a C-terminal linker-subunit moiety and a C- and N-terminallinker is

illustrated in the following structure:

The C-terminus carboxylate of a peptide may be converted to an aminefunctional group with a linker-subunit (e.g., a diamine synthon) to forma peptide having an amine functional group on each end of the peptide towhich the remaining linker moiety can be attached. Examples of suchpeptides modified to have a C-terminal amine function group are:

Wherein n=1 to 4.

Many diamine C-terminal linker-subunits can be conveniently derived froma solid phase resin:

The following resins (R) are commercially available from Nova Biochem:

In some cases, the following linker-subunits may be employed as spacergroups at the N-terminal amine functional group:

wherein “Base” is a purine or pyrimidine base (“Ad”=adenosine,“Gu”=guanosine, “Yh”=thymine, “Cy”=cytosine) and “LG” is a leaving groupsuch as OH, activated ester, halide, or anhydride.

Additionally, αN-alkylated amino acids may be employed, as well as aminoacids having amine-containing side chains (such as Lys and Orn) in whichthe amine has been acylated or alkylated as in the following examples:

wherein n is an integer from 0 to 3, R is any aliphatic or aromaticgroup, and LG is a leaving group such as OH, activated ester, halide,and anhydride.

Still more linker-subunits include the following:

wherein n is independently 1 or 2, R is any aliphatic or aromatic group,and LG is a leaving group such as OH, activated ester, halide, andanhydride.

Examples of linker moieties that are useful when following an amide bondconstruction strategy in which a peptide molecule has two terminal aminegroups include the following:

wherein each m is independently an integer from 1 to 4, n isindependently an integer from 0 to 4 inclusive, LG is a leaving group,and R′ or R″ are independently hydrogen or a chemical protecting group.

A linker moiety also may have branch points for attachment of more thantwo chelates. For example, when following an amide bond formationstrategy, a linker that includes a carbonyl with a leaving group LG (forexample, a carboxylic acid or an activated ester) and three or moreprotected amines can be reacted with a peptide amine to create amolecule with three or more terminal amines. The followingcarbonyl-based linker reagents may be appropriate for introducing threeor more amine functional groups:

wherein LG is a leaving group (e.g., —OH, activated ester such aspentafluorophenol (Pfp), N-hydroxysuccinimide (NHS),N-hydroxysulfosuccinimide sodium salt (NHSS), 2-thioxothiazolidin-1yl,or hydroxybenzotriazole (HBT) and R¹ and R² are preferably independentlyhydrogen or a chemical protecting group (e.g., Boc, Fmoc, CBZ, t-butyl,benzyl, or allyl).

In other embodiments, an amine functional group at the N-terminus of apeptide may be converted to an N-terminus carboxylate functional groupby reaction with a cyclic acid anhydride (linker-subunit moiety) therebyproducing a modified peptide with a N-terminal carboxylate functionalgroup:

Examples of other linker-subunits that can be used to convert anN-terminal amine to a carboxylate functional group include:

wherein R is any aliphatic or aromatic group.

Subsequently, both terminal carboxylates in the above examples may besimultaneously reacted with an amino group on a linker moiety as shownbelow to form a precursor MR imaging agent. In this example, theprecursor MR imaging agent is a peptide molecule derivatized withlinkers at both termnini thru amide bonds:

Specific examples of additional linker moieties useful for producingprecursor MR imagining agents terminating with two carboxylates are:

wherein each m is independently 1 to 4 inclusive, n is independently 0to 4 inclusive (e.g., n=1 or 2), and R is hydrogen or an appropriatechemical protecting group, such as methyl, ethyl, benzyl, or t-butyl. Inthese examples, following attachment of the linker-subunits, theprotecting groups can be removed and chelating or precursor chelatingmoieties can be attached through standard methods, for example, amidebond formation.

When following an amide bond construction strategy in which a peptidemolecule is terminated with two carboxylates, the following linkerreagents may be appropriate to introduce three or more amine functionalgroups:

wherein R¹ and R² are independently hydrogen or a chemical protectinggroup such as OS, Boc, Fmoc, CBZ, tbutyl, benzyl, or allyl.

Linker strategies that involve formation of amide bonds are usefulbecause they typically are compatible with the protecting groups on thepeptide. As mentioned above, the peptide, linker, and linker-subunitsmay be covalently attached to each other by formation of other bondtypes (nucleophilic displacement, reductive amination and thioureaformation, for example).

The linkers may also have effects on the properties of the contrastagents such as affinity, pharnacokinetic properties, stability in vivo,and relaxivity.

Alternatively, a covalent conjugate that includes both a linker moietyand a chelating or chelating precursor moiety can be reacted directlywith a peptide with appropriate terminal functionality. One example ofsuch a covalent conjugate capable of reacting with terminal carboxylategroups follows:

wherein n=1 to 4, and R¹, R², R³, R⁴, and R⁵ are independently anacetate group, acetamide group, or an acetoxy group.

Another example of such a covalent conjugate has the followingstructure:

wherein R¹, R², R³, R⁴, and R⁵ are independently an acetate group,acetamide group, or an acetoxy group.

An example of a covalent conjugate useful for converting a modifiedpeptide with carboxylate functional groups at the two termini to aprecursor imaging agent has the following structure:

Examples of covalent conjugates capable of reacting with aminefunctional groups on a modified peptide are:

wherein LG is a leaving group, n=1 to 4, and R¹, R², R³, R⁴, and R⁵ areindependently an acetate group, acetamide group, or an acetoxy group,and

wherein LG is a leaving group, wherein R¹, R², R³, R⁴, and R⁵ areindependently an acetate group, acetamide group, or an acetoxy group.

A particularly useful covalent conjugate for synthesizing a multimercontrast agent has the following structure, hereinafter “Synthon #1”:

Another embodiment of a covalent conjugate useful for synthesizing amultimer has the following structure, hereinafter “Synthon #2”:

In the following example of an MRI contrast agent of the invention thatincludes a peptide as outlined above, the effect of the N-terminuslinker on the relaxivity of MRI contrast agents is illustrated:

In this example, “chelate” refers to bb-DTPA-Gd(III).

The “Linker-subunit” above was varied with the following results(relaxivities per Gd(III) ion were determined at 20 MHz and 35° C.,units are mM⁻¹s⁻¹):

Relax- Relaxivity Fibrin N-terminal Linker- ivity Fibrin DD(E) Affinitysubunit PBS (10 mg/mL) Structure 15 Ki = 4.0 μM

11.7 18.7 Structure 16 Ki = 3.4 μM

12.6 29.5 Structure 32 (direct bond) 12.5 21.6 Ki = 4.7 μM

As shown above, structures 15 and 16 are similar to 32 except fordifferent N-terminal linker-subunits. The experimental results show thata linker can affect the relaxivity, as well as other characteristics ofa contrast agent of the invention.

Chelating Moieties and Reagents

Chelating moieties are chelating ligands complexed with metal ions.These chelating moieties contain a synthetic moiety capable of forming apoint of attachment to the linker, linker-subunit, and/or modifiedpeptide. One or more chelating moieties may be covalently conjugated tothe functional group at each terminus of the modified peptide. In oneembodiment, the chelate is attached to a linker-subunit. In anotherembodiment, the chelate is attached to a linker moiety. In otherembodiments, the chelate may be conjugated with a linker moiety to forma covalent conjugate before attaching the covalent conjugate to themodified peptide.

Precursor chelating moieties are chelating ligands that have not beencomplexed with metal ions. Chelating ligands may have protecting groupsor may be precursors to chelating ligands. Precursor chelating moietieshave a synthetic moiety capable of forming a point of attachment to thelinker, linker-subunit, and/or modified peptide. Precursor chelatingmoieties can be converted into chelating moieties by complexing with ametal ion. One or more precursor chelate moieties may be covalentlyconjugated to the functional group at each terminus of the modifiedpeptide. In one embodiment, the precursor chelate can be attached to alinker-subunit. In another embodiment, the precursor chelate is attachedto a linker moiety. In other embodiments, the precursor chelate may beconjugated with a linker moiety to form a covalent conjugate beforeattaching the covalent conjugate to the modified peptide.

Precursor chelate moieties and chelate moieties according to theinvention can have any of the following structures:

wherein X is a heteroatom electron-donating group capable ofcoordinating a metal cation, such as O⁻, OH, NH₂, OPO₃ ²⁻, NHR, or OR,wherein R is any aliphatic group; R¹ is an uncharged chemical moiety,selected from hydrogen, any aliphatic, alkyl group, or cycloalkyl group,or uncharged substituted versions thereof (e.g. alcohols); and Y is asynthetic moiety (e.g., capable of forming a point of attachment, orbeing the point of attachment, to the functional group of the modifiedpeptide, linker, and/or linker-subunit either directly or with anintervening carbonyl, methylene, methylene-oxygen, thiocarbonyl).Moieties with (chelate moiety) or without (precursor chelate moiety) acoordinated metal ion may be used.

A variety of chelating ligands may be used in contrast agents of theinvention. Such chelating ligands include, but are not limited to,derivatives of DTPA, DOTA, NOTA, and DO3A. For MRI, metal chelates suchas gadolinium diethylenetriaminepentaacetate (DTPA.Gd), gadoliniumtetraamine 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetate(DOTA.Gd) and gadolinium 1,4,7,10-tetraazacyclododecane-1,4,7-triacetate(DO3A.Gd) are particularly useful. Particularly useful chelates includebb(CO)DTPA.Gd. Other metals may be substituted for Gd(III) in MRIapplications.

Examples of functionalized chelates that have been synthesized for thepurpose of preparing multimeric chelates include pNCS-Bz-DTPA [Martin,V., et al. Bioconjugate Chem. 6,616-23, 1995] andGd(4-NCS-phenyl)-amino-carbonylmethyl-DO3A [Ramachandran, R. et al.,Invest. Rad. 1998, 33(11), 779-797]. For optimal relaxivity propertieswhen bound to a target, it is frequently desirable to minimize chelatemotion, and hence, a minimal number of covalent bonds linking the targetto the chelating ligand are desirable. Below is an example of a reagentthat includes a chelating ligand with a backbone carbonyl group forconnecting to an amine functional group or a linker-subunit or linker,wherein “LG” is a leaving group (e.g., an activated ester) and Rrepresents a group which may be easily cleaved to form O⁻ (—OtBu, e.g. acarboxylate ester) thereby forming a carboxylate with the neighboringcarbonyl group:

It has been found that the chemical motif of a carbonyl immediatelyadjacent to a chelating ligand constitutes a class of high relaxivityMRI contrast agents.

The present invention also relates to intermediates useful in thesynthesis of contrast agents for MRI according to the followingformulae:

wherein, R¹, R², R³, R⁴, and R⁵ may be any protected or unprotectedacetyl ligand suitable for forming a chelate of a paramagnetic metalwith an appropriate formation constant, including the following:

wherein P is any protecting group, including benzyl and tert-butylgroups. LG is a “leaving group” and represents —OH and ester formsthereof including, NHS esters, pentafluorophenol, and other activatedesters.

In a particularly useful embodiment, LG is —OH, and R¹, R², R³, R⁴, andR⁵ are CH₂CO₂O^(t)Bu, hereinafter “Synthon #3”, and has the followingstructure:

Alternatively, the following reagent may be used in the synthesis ofcontrast agents of the invention:

(vide Syn. Comm. 30, 3755 (2000).)

The present invention also provides methods of manufacturing compounds.In particular, a novel oxidation reaction permits the facile preparationof Synthon #3, a preferred embodiment of a chelating ligand. Thesynthesis of Synthon #3 can be achieved through two different syntheticroutes, both commencing with hydroxymethyl-diethylenetriamine. One routeinvolves a two synthetic step sequence (alkylation, followed byoxidation) and the other involves a six-step process (protection,oxidation, esterification, deprotection, alkylation and hydrogenolysis).Both produce Synthon #3 in high chemical and optical purity (videExamples, below).

U.S. Pat. No. 5,637,759 discloses a synthesis of Synthon #1 from2,3-diaminopropionic acid and aza-lysine by a selective hydrolysisprotocol with sodium thiophenoxide. The method disclosed herein avoidsthe use of this toxic reagent.

Synthesis of Contrast Agents

Synthesis of the peptide-based contrast agents may be carried out in thefollowing steps. First, a targeting peptide can be synthesized with orwithout a C-terminal linker-subunit, typically using solid phase peptidesynthesis. For cyclic peptides described herein, a protected linearpeptide may be cyclized in solution or on the resin. Unprotected peptidemay also be cyclized in solution or on resin. A C-terminallinker-subunit may be conveniently derived from the solid phasesynthesis resin and an N-terminus linker or N-terminal linker-subunitcan be coupled to the peptide during the solid phase synthesis.Typically, following cyclization, the linker-subunit-chelate precursormoieties were coupled to the peptide. Protecting groups were removed toprovide the ligand precursors, and then chelates were prepared.Radionuclide compounds of this invention were prepared from ligandprecursors using commercially available radionuclides (for example,^(99m)Tc from Nycomed Amersham Boston cat. #RX-290195, ¹¹¹In from NENLife Science Products cat. # NEZ304, or ¹⁵³Gd from NEN Life ScienceProducts cat. # NEZ142) by reaction in aqueous media, typically at pH4-6 for 1 hour. In the case of optical contrast agents, an organic dyemay be substituted for a chelate precursor.

Structure of Contrast Agents:

Properties of Contrast Agents

Compounds of this invention can be more stable with respect todegradation by endogenous enzymes than the parent peptide (i.e., thepeptide without any attached chelates), a peptide with one or morechelates attached to the N-terminus, or a peptide with one or morechelates attached to the C-terminus. To estimate in vivo stability, testcompounds can be incubated with rat liver homogenates. After selectedintervals, the reactions can be quenched and centrifuged, and thesupernatant can be analyzed by liquid chromatography-mass spectrometryto quantitate the amount of compound remaining.

Compounds of the invention also can bind a target such as human serumalbumin or fibrin. For example, at least 10% (e.g., at least 50%, 80%,90%, 92%, 94%, or 96%) of the contrast agent can be, bound to thedesired target at physiologically relevant concentrations of drug andtarget. The extent of binding of a contrast agent to a target, such asHSA or fibrin, can be assessed by a variety of equilibrium bindingmethods. For example, binding to HSA can be measured by ultrafiltration.For measuring binding to fibrin, a fibrin clot may be formed in a wellof a microtiter plate and contacted with the targeting group. After anincubation time sufficient to establish equilibrium, the supernatant isremoved by aspiration (the insoluble fibrin remains bound as a gelledclot to the bottom of the well). The concentration of unbound targetinggroup in the supernatant is then measured. In both methodologies, theconcentration of bound contrast agent is determined as the differencebetween the total targeting group concentration initially present andthe unbound targeting group concentration following the binding assay.The bound fraction is the concentration of bound targeting group dividedby the concentration of total targeting group.

Compounds of the invention can exhibit high relaxivity as a result oftarget binding (e.g., to fibrin), which can lead to better imageresolution. The increase in relaxivity upon binding is typically1.5-fold or more (e.g., at least a 2, 3, 4, 5, 6, 7, 8, 9, or 10 foldincrease in relaxivity). Targeted contrast agents having 7-8 fold, 9-10fold, or even greater than 10 fold increases in relaxivity areparticularly useful. Typically, relaxivity is measured using an NMRspectrometer. The preferred relaxivity of an MRI contrast agent at 20MHz and 37° C. is at least 10 mM-1s-1 per paramagnetic metal ion (e.g.,at least 15, 20, 25, 30, 35, 40, or 60 mM-1s-1 per paramagnetic metalion. Contrast agents having a relaxivity greater than 60 mM-1s-1 at 20MHz and 37° C. are particularly useful.

As described herein, targeted contrast agents can show an increase inclot uptake. Specificity of uptake of fibrin-targeted agents can bedetermined by comparing the uptake of the agent by blood clots to theuptake by blood. See Example 11 for more details. The specificity offibrin-targeted contrast agents also can be demonstrated using MRI andobserving enhancement of clot signal.

Use of Peptides and Contrast Agents of the Invention

Peptides of the invention can be used to improve therapies for treatingthromboembolic disease. Current thrombolytic therapy has limitations,including a significant risk of bleeding, failure to restore blood flow,thrombotic reocclusion after cessation of therapy, and a lag betweeninitiation of therapy and clot lysis. An improved therapeutic index canbe achieved by conjugating a fibrin targeting peptide of the inventionto a thrombolytic agent (e.g., a protein thrombolytic such asplasminogen activators of human or bacterial origin). Such conjugatescan activate plasminogen locally or increase endogenous levels of tPA.For example, a fibrin targeting peptide can be conjugated to humanplasminogen activators including recombinant tissue type plasminogenactivator (tPA), prourokinase and urokinase (both single and two chainforms), bacterium derived plasminogen activator including streptokinase,staphylokinase, and animal derived plasminogen activators, includingvampire bat plasminogen activator. In addition, fibrin targetingpeptides can be conjugated to fibrinolytics such as copperhead snakefibrolase, which exhibits direct fibrinolytic activity. Such enzymes andproteins can be obtained commercially, extracted from natural sources ortissues, or prepared recombinantly.

The compositions of the invention can be linked or fused in known ways,using the same type of linkers discussed above with respect toconstructing MRI contrast agents. Conjugation to a protein can beachieved by standard chemical techniques including the formation ofamide, ester, disulfide, and thioether bonds. For example, a fibrinbinding peptide can be covalently linked, either directly or through alinker, to a protein by forming an amide bond between the fibrin bindingpeptide or the linker and the lysine residues on the surface of theprotein. These surface lysine residues are usually distant from theenzyme's catalytic site. Therefore, the tethered moieties do notinterfere with the enzyme's catalytic activity. Multiple ligation can beachieved in a single step. The ratio of the fibrin targeting peptide tothe thrombolytic or fibrinolytic agent can be controlled by adjustingthe stoichiometry of the ligation chemistry. Multiple ligation isparticularly useful in the case of a moderately strong fibrin bindingligand because higher binding affinity can be realized through the socalled “avidity” effect. In particular, a coupling agent or an activatedester can be used to achieve amide bond formation between the lysine andthe fibrin binding moiety or the linker. The below scheme shows anexample of a hybrid molecule formed by chemical ligation of urokinase tomultiple fibrin binding peptides via linker moieties. The number ofsurface lysine residues and the number of fibrin binding molecules areillustrative. Alternatively, the fibrin targeting peptide can beincorporated into the hybrid molecule using recombinant DNA technology.

In some embodiments, peptides of the invention can be linked to athrombolytic agent with a linker encompassing an enzymatic cleavagesite, e.g., an enzymatic cleavage site normally cleaved by enzymes inthe coagulation cascade, such as Factor Xa, thrombin, or plasmincleavage sites, etc. The thrombolytic agent is not activated until it iscleaved from the clot binding compositions of the invention at the siteof the clot, the risk of unwanted bleeding events at sites distant fromthe clot would be minimized. Furthermore, thrombolytic moieties can belinked to a peptide-targeted multimeric contrast agent such that a clotcan be identified, imaged and dissolved.

Contrast agents prepared according to the disclosures herein may be usedin the same manner as conventional MRI contrast agents and are usefulfor the diagnosis of deep vein thrombosis, pulmonary embolus, coronarythrombosis, carotid and intracranial thrombosis, atrial and ventricularthrombi, aortic arch thrombi, and high risk plaque. When imaging athrombus, certain MR techniques and pulse sequences may be preferred toenhance the contrast of the thrombus compared to the background bloodand tissues. These techniques include, but are not limited to, blackblood angiography sequences that seek to make blood dark, such as fastspin echo sequences and flow-spoiled gradient echo sequences. Thesemethods also include flow independent techniques that enhance thedifference in contrast due to the T1 difference betweencontrast-enhanced thrombus and blood and tissue, such asinversion-recovery prepared or saturation-recovery prepared sequencesthat will increase the contrast between thrombus and background tissues.Methods of preparation for T2 techniques may also prove useful. Finally,preparations for magnetization transfer techniques may also improvecontrast with agents of the invention.

Compositions of the invention, including peptides, peptides conjugatedto thrombolytics, and peptide-targeted multimeric contrast agents, canbe formulated as a pharmaceutical composition in accordance with routineprocedures. As used herein, the compounds of the invention can includepharmaceutically acceptable derivatives thereof. “Pharmaceuticallyacceptable” means that the compound or composition can be administeredto an animal without unacceptable adverse effects. A “pharmaceuticallyacceptable derivative” means any pharmaceutically acceptable salt,ester, salt of an ester, or other derivative of a compound of thisinvention that, upon administration to a recipient, is capable ofproviding (directly or indirectly) a compound of this invention or anactive metabolite or residue thereof. Other derivatives are those thatincrease the bioavailability of the compounds of this invention whensuch compounds are administered to a mammal (e.g., by allowing an orallyadministered compound to be more readily absorbed into the blood) orwhich enhance delivery of the parent compound to a biologicalcompartment (e.g., the brain or lymphatic system) thereby increasing theexposure relative to the parent species. Pharmaceutically acceptablesalts of the compounds of this invention include counter ions derivedfrom pharmaceutically acceptable inorganic and organic acids and basesknown in the art.

Pharmaceutical compositions of the invention can be administered by anyroute, including both oral and parenteral administration. Parenteraladministration includes, but is not limited to, subcutaneous,intravenous, intraarterial, interstitial, intrathecal, and intracavityadministration. When administration is intravenous, pharmaceuticalcompositions may be given as a bolus, as two or more doses separated intime, or as a constant or non-linear flow infusion. Thus, compositionsof the invention can be formulated for any route of administration.

Typically, compositions for intravenous administration are solutions insterile isotonic aqueous buffer. Where necessary, the composition mayalso include a solubilizing agent, a stabilizing agent, and a localanesthetic such as lidocaine to ease pain at the site of the injection.Generally, the ingredients will be supplied either separately, e.g. in akit, or mixed together in a unit dosage form, for example, as a drylyophilized powder or water free concentrate. The composition may bestored in a hermetically sealed container such as an ampule or sachetteindicating the quantity of active agent in activity units. Where thecomposition is administered by infusion, it can be dispensed with aninfusion bottle containing sterile pharmaceutical grade “water forinjection,” saline, or other suitable intravenous fluids. Where thecomposition is to be administered by injection, an ampule of sterilewater for injection or saline may be provided so that the ingredientsmay be mixed prior to administration. Pharmaceutical compositions ofthis invention comprise the compounds of the present invention andpharmaceutically acceptable salts thereof, with any pharmaceuticallyacceptable ingredient, excipient, carrier, adjuvant or vehicle.

A contrast agent is preferably administered to the patient in the formof an injectable composition. The method of administering a contrastagent is preferably parenterally, meaning intravenously,intra-arterially, intrathecally, interstitially or intracavitarilly.Pharmaceutical compositions of this invention can be administered tomammals including humans in a manner similar to other diagnostic ortherapeutic agents. The dosage to be administered, and the mode ofadministration will depend on a variety of factors including age,weight, sex, condition of the patient and genetic factors, and willultimately be decided by medical personnel subsequent to experimentaldeterminations of varying dosage followed by imaging as describedherein. In general, dosage required for diagnostic sensitivity ortherapeutic efficacy will range from about 0.001 to 50,000 μg/kg,preferably between 0.01 to 25.0 μg/kg of host body mass. The optimaldose will be determined empirically following the disclosure herein.

With respect to treatment of thrombolytic conditions, the quantity ofmaterial administered will depend on the seriousness of thethromboembolic condition and position and the size of the clot. Theprecise dose to be employed and the mode of administration can bedecided according to the circumstances by the physician supervisingtreatment. In general, dosages of the combined composition/thrombolyticagent conjugate will follow the dosages that are routine for thethrombolytic agent alone, although the improved affinity for fibrin/clotbinding added by the compositions disclosed herein may allow a decreasein the standard thrombolytic dosage. Particular thrombolyticscontemplated for use in this therapy (with examples of dose and methodof administration) are as follows:

Streptokinase 1-3 megaunits over 30 minutes to 3 hrs Anistreplase 30units; 2-5 minute injection tPA (wild-type) 50-150 mg; infusion over upto 6 hrs Two-chain urokinase (40-100 mg); infusion over up to 6 hrsSingle-chain urokinase (scuPA) 3-12 megaunits (30-100 mg; infusion overup to 5 hrs Hybrid plasminogen activators 20-100 mg; injection orinfusion and derivatives Muteins of plasminogen 10-100 mg; injection orinfusion activators

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES

Synthesis, characterization, and use of several high relaxivity contrastagent compositions of the invention will be further illustrated in thefollowing examples. The specific parameters included in the followingexamples are intended to illustrate the practice of the invention, andthey do not in any way limit the scope of the invention. Those skilledin the art will recognize, or be able to ascertain using no more thanroutine experimentation, many equivalents to the specific embodimentsand methods described herein. Such experiments are intended to beencompassed by the scope of the claims.

Example 1 Synthesis of Peptide-Based MR Imaging Agents

The peptide with linker-subunit moiety bound to C-terminus(P-[linker-subunit moiety]). The unprotected peptide was prepared usingstandard Fmoc strategy and a diaminotrityl resin. The peptide wascyclized using thallium trifluoroacetate on the resin or in solution.After being cleaved from the resin, the unprotected peptide was purifiedby RP-HPLC (C-18 column, H₂O/CH₃CN/TFA).

Linker Moiety: To a solution of Boc-Dpr(Boc)-OH.DCHA (1 eq.) andpentafluorophenol (1.2 eq.) in dichloromethane was added PS-carbodiimide(1.2-1.5 eq.). The mixture was shaken for 3-5 h at room temperature.After LC-mass results indicated the reaction was complete, the resin wasremoved by filtration and the solvent was evaporated under reducedpressure to give the crude Linker Moiety (Boc-Dpr(Boc)-OPft(N-α-Boc-N-β-Boc-L-diaminopropionic acid pentafluorophenyl ester,356-128) as a white foam.

Precursor MR Imaging Agent: To a solution of P-[linker-subunit moiety](1 eq.) and the Linker Moiety {Boc-Dpr(Boc)-Opft} (2.2 eq.) in DMF wasadded DIPEA (4-6 eq.). The mixture was stirred overnight at roomtemperature. After LC-mass results indicated the reaction was complete,the solvent was removed under reduced pressure. The crude product wasthen stirred in a mixture of TFA, water and anisole (90%/5%/5%) at roomtemperature for 3 h. Diethyl ether was added and a white precipitateformed, which was purified by RP-HPLC (C-18 column, H₂O/CH₃CN/TFA) togive the Precursor MR Imaging Agent (tetrakisamino-peptide as a whitesolid.

Precursor Chelate Moiety: DOTA GA-Opft To a solution of DOTAGA-OH (1eq.) and pentafluorophenol (1.2 eq.) in dichloromethane was addedPS-carbodiimide (1.2-1.5 eq.). The mixture was shaken for 3-5 h at roomtemperature. After LC-mass results indicated the reaction was complete,the resin was removed by filtration and the solvent was evaporated underreduced pressure to give the crude Precursor Chelate Moiety as a whitefoam.

MR Imaging Agent. To a solution of the Precursor MR Imaging Agent (1eq.) and Precursor Chelate Moiety (4.0 eq.) in DMF was added DIPEA (4.0eq.). The mixture was stirred overnight at room temperature. AfterLC-mass results indicated the reaction was complete, the solvent wasremoved under reduced pressure.

The crude product was then stirred in a mixture of TFA, phenol,methylsulfonic acid, anisole and dichloromethane(90%/2.5%/2.5%/2.5%/2.5%) for 15 min. at room temperature. Diethyl etherwas added and a white precipitate formed and was collected as the crudeproduct.

The crude product was reacted with GdCl₃.H₂O in deionized water to formthe crude MR imaging agent, which was purified using RP-HPLC (C-18column, Ethanol/50 mmol AcONH₄). Appropriate fractions were combined andthe ethanol removed under reduced pressure, and then the combinedfractions were treated with sodium acetate for salt exchange. Afterlyophilization the excess salts were removed using reverse-phasechromatography on a Waters Sep-Pak® C-18 cartridge with water andethanol:water (50:50) eluants. Appropriate fractions were combined, theethanol removed under reduced pressure, and the solution was lyophilizedto give the desired peptide MR imaging agent as a white solid.

Similar methods were used to synthesize other MR imaging agents.

Example 2 Methods for Synthesis of A Chelate Precursor Moiety (Synthon#3)

Method A for Synthesis of Synthon #3

Hydroxymethyl-diethylenetriamine of the indicated stereochemistrytrihydrochloride (25.15 g) (optically pure starting material: Syn. Comm.29(14), 2377-2391 (1999), racemic starting material: Coll. Czech. Chem.Comm. 34, 630-634 (1969)) was dissolved in a deionized water/1,4-dioxanemixture and the pH of the solution was adjusted to between 8 and 9 withaqueous sodium hydroxide. Di-tert-butyl dicarbonate (3.5 equiv.) wasdissolved in dioxane and added between 10 and 20° C. The reactionmixture was stirred between 12 and 20 hours at room temperature. Thereaction mixture was then diluted with water, and extracted with ethylacetate. The organic extract was extracted sequentially with water,saturated sodium bicarbonate, and saturated sodium chloride solutions.The organic extract was dried over sodium sulfate, filtered, andconcentrated under in vacuo to provide an oil which was purified bysilica gel chromatography with a mixture of ethyl acetate:hexane. Thetotal yield of purified product was 30.11 g. ¹H NMR (300 MHz): 5.18 (d,J=7.9 Hz, 1H), 4.76 (bs, 1H), 3.8-3.0 (m, 10H), 1.47-1.42 (2s, 27H). MS(m/Z): 456.4 [M+Na]⁺.

Step b—Oxidation of Hydroxyl Group

[based on the oxidation procedure disclosed in Zhao et al. J. Org. Chem.64, 2564-2566 (1999)]

BOC-protected triamine (29.94 g) was dissolved in acetonitrile.Phosphate buffer, consisting of 21.6 g NaH₂PO₄, 21.6 g Na₂HPO₄ andenough deionized water to produce a 500 mL volume, was added (300 mL),followed by 2,2,6,6-tetramethylpiperidinyl-1-oxy (TEMPO) (0.07 equiv.).The mixture was stirred vigorously and warmed to 35° C. Sodium chlorite(2.0 equiv.) was dissolved in deionized water (100 mg/ml). The sodiumchlorite solution and bleach (0.02 equiv., approx. 0.25% aqueous sodiumhypochlorite) were added while maintaining a constant temperature. Afteraddition of oxidant, the reaction was stirred for 24 hours. AdditionalTEMPO (0.07 equiv.) was added and the reaction mixture was stirred for24 hours. The reaction was cooled to room temperature. Water was addedand the pH was adjusted to 8 with 2.0 N aqueous NaOH. A cold solution ofaqueous sodium sulfite was added (300 mL) while maintaining a constantpH. The solution was extracted with a small volume of methyl tert-butylether and set aside. The aqueous layer was acidified to pH 3-4 with 2.0N aqueous HCl and extracted with two small volumes of methyl tert-butylether. The organic extract was combined with the one previously setaside and concentrated in vacuo. The product was used withoutpurification in Step 3 below. ¹H NMR (300 MHz): 5.8 (bs, 1H), 5.3 (m,1H), 4.4 (M, 1H), 3.6-3.2 (m, 6H), 1.47-1.43 (2s, 27H). MS (m/Z): 470.2[M+Na]⁺.

Step c—Benzyl-Protection of Carboxylic acid

The carboxylic acid starting material (178 g) was dissolved in dry DMF.Cesium carbonate (2.0 equiv.) was added, and the solution was stirredfor 30 minutes. Benzyl bromide (1.1 eq.) was added dropwise at roomtemperature. The reaction mixture was stirred under an inert atmospherefor 18 hours. The reaction mixture was diluted with water and extracttwice with ethyl acetate. The organic layers were combined and washedsequentially with saturated sodium bicarbonate and sodium chloridesolutions. The organic layer was concentrated to an oil (270 g) whichwas purified by silica gel chromatography using ethyl acetate:hexane. ¹HNMR (300 MHz): 7.3 (s, 5H), 5.6 and 5.15 (2bs, 1H), 5.1 (s, 2H), 4.5(bs, 1H), 4.0-4,1 (m, 1H), 3.5-3.2 (m, 6H), 1.45 and 1.4 (2s, 27H). MS(m/Z): 560.3 [M+Na]⁺.

Steps d and e—Deprotection of Amines and Alkylation

The BOC-protected triamine (250 g) was stirred in a solution of 1:1acetonitrile:4 N aqueous HCl and allowed to stir for approximately 2.0hours. The acetonitrile was removed in vacuo and the remaining solutionwas lyophilized to provide a residue that was immediately dissolved inDMF and diisopropylamine (a sufficient amount to raise the pH to 8).tert-Butyl bromoacetate was added (12.0 equiv.). After the addition wascomplete, the reaction mixture was warmed to 50° C. and stirred for 18hours. Upon completion of reaction the volume of the reaction mixturewas doubled by the addition of water, after which it was extracted twicewith ethyl acetate. The organic extracts were combined and washedsequentially with water, saturated sodium bicarbonate, and saturatedsodium chloride solutions. The organic solution was concentrated invacuo to an oil which was purified by silica gel chromatography usingethyl acetate:hexane. The total yield of purified product was 190 g. MS(m/Z): 809.5 [M+Na]⁺.

Step f—Deprotection of Carboxylate

A stainless steel reactor was charged with benzyl ester (157 g), 10%palladium on carbon (19.8 g) and ethyl acetate and hydrogenated at 45psi for 12 hours. Filtration through Celite® and concentration in vacuogave an oil. The oil was dissolved in ethyl acetate/hexanes and purifiedby silica gel chromatography to provide the DTPA carboxylic acidpenta-tert-butyl ester(yield: 82%). MS (m/Z): 719.5 [MH]⁺. When thisreagent is used in the synthesis of contrast agents using other chiralelements, no diastereomers are observed and therefore it is concludedthat this material is essentially optically pure to the limit ofdetection by ordinary proton NMR.

Method B for Synthesis of Synthon #3

Alcohol (see Syn. Comm. 29(14), 2377-2391 (1999) for a synthesis fromhydroxymethyl-diethylenetriamine) starting material (105.0 g),acetonitrile (1.0 L), phosphate buffer (1.0 L, prepared by dissolving100 mg of NaH₂PO₄ and 10 mg of Na₂HPO₄ into 1.0 mL of water and thenadjusting the pH to 4.5 with H₃PO₄) and TEMPO (7.0 g) were combined andwarmed to between 45 and 50° C. A solution consisting of sodium chlorite(29.8 g dissolved in 298 mL of water) and sodium hypochlorite (744 μL)was added to the solution while maintaining a temperature of 45 to 50°C. The reaction mixture was stirred vigorously for 4 to 10 hours. Thereaction mixture was cooled to room temperature and two layers wereseparated. The organic layer was isolated and combined with saturatedaqueous sodium chloride and stirred for 15 minutes. The organic layerwas isolated and concentrated in vacuo to give an oil (171 g) which waspurified by column chromatography using hexane:isopropanol with 0.1%triethylamine throughout to provide 81 g of enriched product as an oil.MS (m/Z): 719.5 [MH]⁺. The optical purity of material produced by thismethod did not differ from that above.

Example 3 Resins for Solid Phase Synthesis of Modified Peptides withC-terminal Amine Functional Groups

The peptides have been prepared by solid-phase synthesis. In solid-phasesynthesis, the linkers and resins are selected depending on the type ofthe peptides to be synthesized (e.g., the functional group required atthe C-terminus, the protected or unprotected peptide) and the syntheticmethod to be used (e.g. Fmoc or BOC chemistry, manual or automatedsynthesis, the continuous flow or batch reactor). For example, in thesynthesis of a peptide with a carboxylic group at the C-terminus, aprotected-amino acid is attached to the different resins such as HMPBresins, 2-chlorotritylchloride resin and SUSRIN resin. On the otherhand, in the synthesis of a peptide with an amino group at theC-terminus, a diamine can be attached to a trityl resin. The polystyrene(PS) resins can be used for batch synthesis, while polyethyleneglycol(PEG) modified resins are suitable for continuous flow and batchsynthesis. Many trityl PS resins including 1,3-bis-(aminomethyl)-benzenetrityl PS resin are commercially available. If the required trityl PSresin is not available, a similar procedure as described for PEG resinscan be used to attach a diamine to a trityl PS resin.

The synthesis of 1,3-bis-(aminomethyl)-benzene trityl resin is discussedas an example. Other diamines can be attached to trityl resin in asimilar manner.

Preparation of 1,3-bis-(aminomethyl)-benzene trityl PEG resin

First, trityl alcohol resin (25 g, NovaSyn TGT resin, NovaBiochem) wasplaced in a funnel and washed sequentially with DMF, CH₂Cl₂, andtoluene. After removing all solvent, the material was transferred to aflask equipped with a reflux condenser. Toluene (250 mL) and acetylchloride (25 mL) were added and the slurry was heated to 70° C. andstirred for 1.0 hour. An additional portion of acetyl chloride (25 ml)was added and the slurry was stirred for 2.0 hours at 70° C. The slurrywas filtered and the resin washed sequentially with toluene and CH₂Cl₂.

Second, freshly prepared trityl chloride resin and THF (250 mL) wasplaced in a flask. To the slurry was added 1,3-bis-(aminomethyl)-benzene(10 eq., based on a resin substitution of 0.23 mmol/g) and the mixturewas stirred for 18.0 hours at room temperature. The slurry was filteredand the resin was washed sequentially with water, DMF, CH₂Cl₂, methanol,and CH₂Cl₂. The resin was dried under vacuo (room temperature, 1-5 mmHg) to a constant weight (25.4 g). The substitution stoichiometry wasconducted using a quantitative ninhydrin procedure.

Example 4 Synthesis of Covalent Conjugates (synthon #1, #2, #4, and #5)

2,3-Bis-tert-butoxycarbonylamino propionic acid benzyl ester

A solution of cesium carbonate (6.5 g) and water was added to2,3-bis-tert-butoxycarbonylamino propionic acid (3.04 g) in acetonitrile(25 ml). The mixture was stirred for 40 minutes at room temperature. Thesolvent was removed under vacuum. DMF (50 ml) was added to the solidresidue. A solution of benzyl bromide (1.43 ml) and DMF (5.0 ml) wasadded over 15 minutes at room temperature. The mixture was stirred for18 hours, and then the mixture was diluted with ethyl acetate (100 ml)and water (50 ml) and stirred for 15 minutes. The layers were separatedand the organic layer was dried over sodium sulfate. The mixture wasfiltered, and the filtrate was concentrated under vacuum to give an oil(3.6 g). The oil was purified by column chromatography with ethylacetate/hexane to provide an oil (1.8 g). MS (m/Z): [M+Na]⁺=417. ¹H NMR(300 MHz): 1.4 (s, 9H), 2.4 (m, 2H), 4.4 (m, 1H), 4.8 (m, 1H), 6.5 (m,1H), 7.3 (m, 5H).

2,3-Diamino-propionic acid trihydrochloride

A solution of 2,3-bis-tert-butoxycarbonylamino propionic acid benzylester (1.8 g), 4N aqueous HCl (40 ml), and acetonitrile (50 ml) wasstirred for 18 hours at room temperature. The solvent was removed undervacuum, and the mixture was diluted with ethyl acetate (100 ml) andwater (50 ml) and stirred for 15 minutes. The layers were separated andthe aqueous layer was evaporated to dryness (1.23 g of a foam/syrup). ¹HNMR (300 MHz): 3.2-3.5 (m, 2H), 4.2-4.35 (m, 1H), 5.13-5.25 (q, 2H,J=11.8, 3.1 Hz).

2,3-Bis-carboxy-DTPA propionic acid benzyl ester

A solution of 2,3-diamino-propionic acid trihydrochloride (6.65 g),bb(CO)DfPE (40.0 g, “Synthon 3”), HOBt (8.5 g), DIC (9.0 ml),diisopropylethylamine (15.0 ml), CH₂Cl₂ (400 ml) and DMF (200 ml) wasstirred for 48 hours. The mixture was diluted with methylene chloride(500 ml) and water (250 ml), and then stirred for 15 minutes. The layerswere separated and the organic layer was washed with saturated sodiumcarbonate (250 ml), saturated sodium chloride (250 ml) and dried oversodium sulfate. The mixture was filtered and the filtrate wasconcentrated in vacuo to obtain an oil. The oil was purified by silicagel chromatography using ethyl acetate and hexanes to obtain 28.0 g ofmaterial. MS (m/Z): [M+2]⁺=798; [M+1]⁺=1595.

2,3-Bis-carboxy-DTPA propionic acid

Hydrogenation of 2,3-bis-carboxy-DTPA propionic acid benzyl ester (17.0g) was performed in ethyl acetate/triethylamine (10:3) using 10%palladium on carbon (6.0 g) catalyst for 24 hours at 50 psi. The vesselwas purged with nitrogen and the mixture was filtered through Celite®,and concentrated under reduced pressure to give 16.0 g of an oil. MS(m/Z): [M+2]⁺=753; [M+1]⁺=1504.

N¹,N³-Bis[2-(bis-tert-butoxycarbonylmethyl-amino)-3-{[2-(bis-tert-butoxycarbonylmethyl-amino)-ethyl]-(Tert-butoxycarbonylmethyl)-amino}-propionamide]-diethylenetriamine

To a solution of diethylenetriamine (3.16 ml), acetonitrile (700 ml),and diisopropylethylamine (10.4 ml) was added pre-reacted (30 min)bb(CO)DTPE (42.0 g, “Synthon 3”), HOBt (7.9 g), EDC (11.2 g) anddiisopropylethylamine (10 ml) in acetonitrile at room temperature. Thereaction was stirred for 16.0 hours and then concentrated under reducedpressure. The oil was combined with ethyl acetate, extracted with waterand saturated aqueous sodium chloride and then concentrated. The oil waspurified by silica gel column chromatography usinghexanes/isopropanol/triethylamine to provide 22.5 g of product. MS(m/Z): [M+H]⁺=1503.

N¹,N³-Bis[2-(bis-tert-butoxycarbonylmethyl-amino)-3-{[2-(bis-tert-butoxycarbopylmethyl-amino)-ethyl]-(tert-butoxycarbonylmethyl)-amino}-propionamide]-N²-(benzyloxycarbonylmethyl)-diethylenetriamine

Benzyl-2-bromoacetate (2.54 g) was added to a solution of the previousamine compound (13.5 g), acetonitrile (200 ml) and sodium carbonate(1.18 g). The mixture was warmed to 60° C. and stirred for 15 hours. Thereaction mixture was cooled to room temperature and concentrated underreduced pressure. Ethyl acetate and water were added, the layers wereseparated. The organic layer was washed with saturated aqueous sodiumchloride, then concentrated under reduced pressure to give an oil (14.5g). MS (m/Z): [M]⁺=1651.

N¹,N³-Bis[2-(bis-tert-butoxycarbonylmethyl-amino)-3-{[2-(bis-tert-butoxycarbonylmethyl-amino)-ethyl]-(tert-butoxycarbonylmethyl)-amino}-propionamide]-N²-(aceticacid)-diethylenetriamine

The above benzyl ester (14 g) was hydrogenated in ethylacetate/triethylamine (49:1) at 50 psi for 16.0 hours in the presence of10% palladium on charcoal (3.5 g). The resulting mixture was filteredthrough Celite®, and concentrated in vacuo to provide 12.08 g of an oil.MS (m/Z): [M+H]⁺=1561.

N¹,N³-Bis-Butoxycarbonyl-Diethylenetriamine

To a solution of diethylenetriamine (2.12 g, 20.6 mmol) andtriethylamine (30.0 g, 41.3 mmol) in THF (100 mL) was added Boc-ON(10.65 g, 43.3 mmol) at room temperature. The mixture was stirred forovernight. To the mixture was added 700 mL of ether and then extractedwith phosphate buffer (pH=3, 100 mmol). The aqueous solution wasbasified to pH=11 and extracted with dichloromethane. The organic layerwas separated and dried over anhydrous sodium sulfate. The salts werefiltered and the solvent was removed under the reduced pressure to givethe indicated compound as a colorless oil (5.55 g). MS (m/Z):[M+H]⁺=304.1.

N¹,N³-Bis-Butoxycarbonyl-N²-(Benzyloxycarbonylethyl)-Diethylenetriamine

To a solution of the compound above (1.0 g, 3.30 mmol) in methanol (40mL) was added benzyl acrylate (1.07 g, 6.59 mmol) at room temperature.The mixture was refluxed for 3 days. The solvent was removed at reducedpressure to give a yellow liquid that contains the indicated compoundand benzyl acrylate. MS (m/Z): [M+H]⁺=466.1.

N²-(Benzyloxycarbonylethyl)-Diethylenetriamine

To a mixture of the compound above and benzyl acrylate (1.0 g) indichloromethane (25 mL) was added TFA (13.8 mL) at room temperature. Themixture was stirred for 3 h at room temperature and then to the mixturewas added 1N HCl and water. The aqueous layer was separated andlyophilized to give the indicated compound (420 mg) as a sticky solid.MS [m/Z]: [M+H]⁺=266.2.

N¹,N³-Bis[2-(Bis-Tert-Butoxycarbonylmethyl-Amino)-3-{[2-(Bis-Tert-Butoxycarbonylmethyl-Amino)-Ethyl]-(Tert-Butoxycarbonylmethyl)-Amino}-Propionamide]-N²-(Benzyloxycarbonylethyl)-Diethylenetriamine

To a solution of the above compound, “Synthon 3”, HOBt anddiisopropylethylamine, in CH₂Cl₂ and DMF is added DIC. The mixture isstirred for 48 h at room temperature. The mixture is diluted withdichloromethane and water, and then stirred for 15 minutes. The layersare separated and the organic layer is washed with saturated sodiumcarbonate, saturated sodium chloride and dried over sodium sulfate. Themixture is filtered and the filtrate is concentrated in vacuo to obtainan oil. The oil is purified by silica gel chromatography using ethylacetate and hexanes to obtain the indicated compound.

N¹,N³-Bis[2-(Bis-Tert-Butoxycarbonylmethyl-Amino)-3-{[2-(Bis-Tert-Butoxycarbonylmethyl-Amino)-Ethyl]-(Tert-ButoxycarboDylmethyl)-Amino}-Propionamide]-N²-(PropionicAcid)-Diethylenetriamine

A hydrogenation vessel is charged with the above compound, 10% palladiumon carbon, ethyl acetate, and triethylamine. The vessel is purged withnitrogen then hydrogen. The mixture is shaken for 24 hours under ahydrogen atmosphere (50 psi). The vessel is purged with nitrogen and themixture is filtered through Celite®, and the filtrate is concentratedunder reduced pressure to give the product as an oil.

Methyl 3-(Benzylamino)-2-((Benzylamino)Methyl)-Proprionate

Methyl-2-(bromomethyl)-acrylate (1.00 g, 5.6 mmol, 1.0 eq.) dissolved inacetonitrile (20 mL) was added drop-wise with stirring at roomtemperature to a solution of benzylamine (1.83 mL, 16.8 mmol, 1.5 eq.)in anhydrous acetonitrile (10 mL). After 16 hours, ether (100 mL) wasadded, and the white solid (benzylamine hydrobromide) was filtered. Thefiltrate was concentrated under reduced pressure to give 1.90 g of acrude oil. The oil was dissolved in ethyl acetate (150 mL) and washedwith H₂O and NaCl brine. The organic layer was dried (MgSO₄), andevaporated under reduced pressure. The resulting clear oil was purifiedby flash chromatography (hexanes:ethyl acetate) to give 1.38 g (79%) ofthe product. ¹H NMR (300 MHz, CDCl₃): δ 1.68 (s, 2H), 2.79-2.90 (m, 5H),3.68 (s, 3H), 3.75 (s, 3H), 7.21-7.32 (m, 10H).

Methyl 3-(Amino)-2-(Aminomethyl)-Proprionate

Methyl 3-(benzylamino)-2-((benzylamino)methyl)-proprionate (2.27 g, 7.3mmol, 1.0 eq) was dissolved in methylene chloride (20 mL) and 4.0 mL ofa 4M solution of HCl in dioxane was added. The solution was stirred atroom temperature for 20 minutes and solvents were then evaporated underreduced pressure to give a white powder that was dissolved in 50 mLMeOH. Catalyst (10% Palladium on carbon catalyst, 750 mg) was added at0° C. under argon and the mixture was shaken at 45 psi H₂ for 18 hours,then filtered through Celite®. The product (1.47 g) was isolatedfollowing a MeOH was and evaporation under reduced pressure. ¹H NMR (300MHz, D₂O): δ 3.25-3.43 (m, 5H), 3.82 (s, 3H).

Methyl 3-(BOC-Amino)-2-(BOC-Aminomethyl)-Proprionate

Diamine (1.44 g) was reacted with di-tert-butyl dicarbonate (3.22 g,14.8 mmol, 1.05 eq.) in dioxane/aqueous Na₂CO₃ at solution 0° C. for 1hour and then room temperature for 18 hours. The mixture was thenacidified (pH=4) with 0.5N KHSO₄ and dioxane was evaporated underreduced pressure. The aqueous portion was extracted with ethyl acetate,dried over MgSO₄ and concentrated under reduced pressure to give a crudeoil that was purified by flash chromatography (hexanes:etbyl acetate) togive 1.86 g (80%) of the desired. NMR (300 MHz, CDCl₃): δ 1.41 (s, 18H),2.66-2.78 (m, 1H), 3.10-3.27 (m, 2H), 3.50-3.62 (m, 2H), 3.68 (s, 3H),5.17-5.27 (bt, 2H).

3-(BOC-Amino)-2-(BOC-Aminomethyl)-Propanoic Acid

Methyl ester (1.50 g) was dissolved in 15 mL of a 2:1 mixture ofTHF/H₂O. LiOH—H₂O (0.95 g) was added at 0° C. The mixture was stirredbetween 0° C. and room temperature for 44 hours. THF was evaporatedunder reduced pressure and the aqueous solution was extracted withEtOAc. The aqueous layer was made acidic (pH=3) with 0.5M aqueous KHSO₄and extracted with EtOAc. Combined organic fractions were washed with 30mL H₂O, and dried (MgSO₄). Solvents were evaporated under reducedpressure to give 1.32 g (92%) of the product. ¹H NMR (300 MHz, CDCl₃): δ1.40 (s, 18H), 2.66 (s, 1H), 3.27-3.47 (m, 4H), 5.42 (s, 1H).

Benzyl 3-(BOC-Amino)-2-(BOC-Aminomethyl)-Propionate

Acid (1.01 g) was reacted at room temperature with benzyl bromide (0.45mL) in anhydrous DMF containing Cs₂CO₃ (2.07 g). DMF was evaporatedunder reduced pressure and the residue was partitioned between H₂O andEtOAc. The organic layer was washed with brine, and dried (MgSO₄).Solvent was evaporated under reduced pressure and the residue purifiedby flash chromatography on silica gel (Hexanes/EtOAc 95:5 to 9:1 to85:15). Yield 1.16 g (90%). ¹H NMR (300 MHz, CDCl₃): δ 1.42 (s, 18H),2.79 (quint, 1H, 5.6 Hz), 3.1-3.3 (m, 2H), 3.5-3.65 (m, 2H), 5.13 (s,2H), 5.15-5.28 (m, 1H), 7.30-7.40 (m, 5H). MS: 431.15 (M+1).

Benzyl 3-Amino-2-Aminomethyl-Propionate Dihydrochloride Salt

Benzyl 3-(BOC-amino)-2-(BOC-aminomethyl)-propionate (1.15 g) wasdissolved in 5 mL 4M HCl solution in dioxane. The mixture was stirred at0° C. for 6 hours and the dioxane was evaporated under reduced pressure.The residue was tritrated with ether and was filtered to give theunpurified diamine dihydrochloride salt (0.81 g) ¹H NMR (300 MHz, MeOD):δ 3.1-3.3 (m, 5H+CHD₂OD), 3.55 (s, dioxane), 5.19 (s, 2H), 5.15-5.28 (m,1H), 7.20-7.40 (m, 5H) MS: 209.00 (M+1)

Benzyl 3-(N-BB(CO)DTPE Carboxamide)-2-(N BB(CO)DTPE Carboxamide)Methyl-Propionate

Acid (2.00 g, 2.8 mmol, 1.5 eq.) was dissolved in 5 mL anhydrousdichloromethane. The diamine (0.26 g), HOAt (0.32 g), anddiisopropylethylamine (0.65 mL) were reacted 0° C. with HATU (0.88 g)for 2 hr. Tris amine resin (0.5 g), isocyanate resin (0.5 g), and HATU(0.42 g) were added and the reaction mixture was stirred for 16 h atroom temperature. The resins were filtered and the filtrate wasevaporated. The residue was partitioned between H₂O and EtOAc. Theorganic layer was washed with H₂O, saturated NaHCO₃, and brine (20 mL),and dried (MgSO₄). Solvent was evaporated under reduced pressure. Theoil was purified by flash chromatography on silica gel(Hexanes/Acetone/Et₃N) and gave product (1.13 g). MS: 1607.95 (M+1) and1629.95 (M+Na).

3-(N-BB(CO)DTPE Carboxamide)-2-(N-BB(CO)DTPE Carboxamide)Methyl-Propionic Acid

Benzyl ester (0.90 g, 0.56 mmol) was dissolved in 30 mL EtOAc and Et₃N(1 mL) was added. 10% Palladium catalyst on carbon (0.50 g) was addedand the mixture was shaken for 15 h under 45 psi Hydrogen. The catalystwas filtered and the solvents evaporated to give the product (0.82 g).MS: 759.55 (M+2H/2)

Example 5 Synthesis of Fibrin-Binding MR Imaging Agents

Peptide Construction: 3.39 g of the 1,3-bis-(aminomethyl)-benzene tritylNovaSyn TGT resin (measured substitution=0.59 mmol) was placed in astandard peptide column and loaded onto a Peptide Synthesizer. Synthesiswas carried out using a standard Fmoc strategy. Capping was carried outafter coupling the first amino acid using acetic anhydride, 6%diisopropylethylamine in DMF. When the synthesis was complete, the resinwas removed from the column and placed in a reaction vessel. The resinwas rinsed once with CH₂Cl₂ and filtered. The resin was then treatedwith 1% trifluoroacetic acid in CH₂Cl₂ and placed on a mechanical shakerfor 10 minutes. The mixture was filtered through the reaction vessel andthe filtrate was collected. The pH of the combined filtrate was adjustedto approximately 8 with triethylamine, and the solution was concentratedunder vacuum to an oil. Following precipitation with water, the whitesolid was filtered and rinsed with water and diethyl ether. The solidwas dried by suction filtration, taken up in DMF and diluted withacetonitrile. The solution was cooled in an ice bath and was treatedwith 1.5 g of thallium trifluoroacetate for 2.0 hours. The pH of thesolution was adjusted with triethylamine to pH 8 and then concentratedunder vacuum. Water was added to the oil and the resulting precipitatewas collected by suction filtration. The solid was washed with water anddiethyl ether to give 2.7 g of crude modified peptide having aC-terminal amine functional group. An example synthesis ofH₂N-Leu-Pro-Cys-Asp-Tyr-Tyr-Gly-Thr-Cys-Bip-Asp-CO—NHCH₂C₆H₄CH₂NH₂ (SEQID NO:21) was confirmed by an observed m/Z of 1792.8 [M+Na]⁺. Themodified peptide was purified by preparative HPLC. Fractions of similarpurity (98-100%) were combined and lyophilized without neutralization.

Modified peptide (3.2 g) and covalent conjugate (Synthon #2 above) (3.95g) were dissolved in dichloromethane. Diisopropylethylamine was addeddropwise until the pH measured 9, and diisopropylcarbodiimide (2 eq.)and HOBt (2 eq.) were added simultaneously to the mixture. Afterstirring 2 minutes, diisopropylethylamine was added dropwise until thepH measured 9. The mixture was stirred at room temperature for twohours. Additional pre-activated Synthon #2 (withdiisopropylcarbodiimide, diisopropylethylamine, and HOBt) was added inone portion. Solvents were removed in vacuo and the residue wasdissolved in ethyl acetate, which was washed sequentially with 0.1 Nhydrochloric acid, saturated sodium bicarbonate, and saturated aqueoussodium chloride. The organic layer was dried over sodium sulfate,filtered, and concentrated under reduced pressure to obtain a lightyellow foam (7.8 g). The foam was dissolved in dichloromethane andpurified by flash chromatography (dichloromethane: methanol eluent) toprovide a white solid (5.6 g). The white solid was stirred in a mixtureof TFA, water, and triethylsilane (90%/5%/5%, 30 ml) at roomtemperature. The mixture was heated to 40° C. and stirred for 2 hours.The solution was concentrated to a volume between 3-5 ml, then cooled toroom temperature. Diethyl ether was added and a white precipitateformed. The mixture was allowed to stir for 10 minutes, and the solidswere collected by filtration and washed with diethyl ether. The solidswere dried under vacuum providing a white solid (4.0 g). The solid wasdissolved in a mixture of water: acetonitrile (20 ml, 4:1 ratio) andpurified by Prep HPLC to yield a white solid, precursor MR imaging agent(1.6 g).

Precursor MR imaging agent (1 g) was reacted with one equivalent ofGdCl₃.6H₂O in distilled deionized water with the pH adjusted to ca. 6 bythe addition of 1 M NaOH. The gadolinium complex was purified by reversephase chromatography (Waters Sep-Pak® C-18) using distilled deionizedwater and 50:50 (v:v) methanol:water eluent. Appropriate fractions werecombined and the methanol removed under reduced pressure at 50° C. andlyophilized to give 811 mg of the MR imaging agent.

Alternatively to the synthesis of 32 presented above, the peptide may becyclized on a resin as illustrated in the following Scheme:

Example 6 MR Imaging Agents Prepared in Analogous Fashion

Each of the following MR imaging agents was synthesized analogously tothe methods described above. Peptide, prepared using standard Fmocstrategy and cyclized using thallium trifluoroacetate, was purified byHPLC and reacted with Synthon #2, diisopropylethylamine,diisopropylcarbodiimide (2 eq.) and HOBt (2 eq.) in dichloromethane.Solvents were removed in vacuo and the residue was dissolved in ethylacetate, which was washed sequentially with 0.1 N hydrochloric acid,saturated sodium bicarbonate, and saturated aqueous sodium chloride. Theorganic layer was dried over sodium sulfate, filtered, and concentratedunder reduced pressure to obtain a foam which was purified if necessaryby flash chromatography or HPLC. The resulting white solid was stirredin a mixture of TFA, water, and triethylsilane (90%/5%/5%, 30 ml) atroom temperature for 2-6 hours. Diethyl ether was added and a whiteprecipitate formed, which was purified by Prep HPLC (CH3CN/H2O/AcONH4)to yield a white solid, precursor MR imaging agent.

Precursor MR imaging agent was reacted with one equivalent of GdCl₃.6H₂Oin deionized water (pH 6, NaOH). The gadolinium chelate was purifiedusing reverse-phase chromatography on a Waters Sep-Pak® C-18 cartridgewith water and methanol:water 50:50 eluant. Appropriate fractions werecombined and the methanol removed under reduced pressure at 50° C. andlyophilized to give the desired MR imaging agent. Table 3 provides massspectrometry data confirming each of the compounds. See the detaileddescription for the structure of each of the compounds.

TABLE 3 MS data of Fibrin-Binding compounds Molecular MS (M + 3H)³⁺/3Compound Weight calcd obsd 4 4016.884 1256.49467 1255.66 5 4025.961259.52 1259.9 6 4108.014 1286.87133 1284.53 7 4307.336 1353.312 1369.348 4076.064 1276.22133 1298.48 9 4233.208 1328.60267 1327.94 10 4221.1991324.59967 1324.57 11 4142.146 1298.24867 1321.13 12 4123.378 1291.992671289.80 13 4325.675 1359.425 1333.3 14 4470.833 1407.811 1407.44 154363.362 1371.98733 1393.99 16 4511.483 1421.361 1444.57 17 4169.1281307.24267 1329.85 18 4503.826 1418.80867 1419.25 19 4387.428 1380.009331379.12 20 4305.369 1352.65633 1351.6 21 4419.473 1390.691 1390.53 224277.356 1343.31867 1341.04 23 4357.829 1370.143 1370.63 24 4443.6441398.748 1398.9 25 4448.209 1400.26967 1400.11 26 4326.731 1359.7771359.9 27 4423.505 1392.035 1392.79 28 4343.375 1365.325 1364.7 294342.387 1364.99567 1364.7 30 4313.366 1355.322 1354.9 31 4342.3871364.99567 1364.8 32 4319.303 1357.301 1357.6

Example 7 Synthesis of Fibrin-Binding Optical Contrast Agents

Each of the following optical contrast agents is synthesized analogouslyto the methods described above. Scheme X shows a general example whereintwo identical optical dyes are added to the same peptide.

5-Carboxytetramethylrhodamine-containing compound (3A)

The peptide 1 (177 mg, 0.10 mmol) and 5-carboxytetramethylrhodaminesuccinimidyl ester A (111 mg, 0.21 mmol) are dissolved indichloromethane (20 mL) and DMF (20 mL). Diisoproopylethylamine is addeddropwise until the pH measures 9. The mixture is stirred for overnightand then the solvents are removed under reduced pressure. The residue ispurified by silica-gel flash column chromatograph(eluants:dichloromethane/methanol) to give compound 2A.

To compound 2A is added a solution of TFA, H₂O and triethylsilane(ratio: 90/5/5, 5 mL). The mixture is shaken for 3 h at roomtemperature, and then the reaction mixture solution is poured into 50 mLof ether to precipitate the crude product. After the solvents areseparated by centrifugation, the crude product is collected and thenpurified using reverse-phase HPLC to obtain compound 3A.

5-Carboxyfluorescein-containing compound (3B)

In a similar procedure as described in the synthesis of 3A, 3B issynthesized using the peptide 1 (177 mg, 0.10 mmol) and5-carboxyfluorescein succinimidyl ester B (99.4 ng, 0.21 mmol).

Texas Red®-X-containing compound (3C)

In a similar procedure as described in the synthesis of 3A, 3C issynthesized using the peptide 1 (177 mg, 0.10 mmol) and Texas Red®-Xsuccinimidyl ester C (172 mg, 0.21 mmol).

Example 8 Measuring Binding of Contrast Agents to Targets

The extent of binding of a contrast agent according to the presentinvention to a target, such as HSA or fibrin, can be assessed by avariety of equilibrium binding methods. For example, binding to HSA canbe measured by ultrafiltration. In a typical binding measurement usingultrafiltration, the contrast agent is mixed with 4.5% weight/volume HSAin a pH 7.4 buffer. The sample is loaded into a commercially availablecentrifugation apparatus equipped is with a 30 kDa molecular weightcutoff filter (Millipore Ultrafree MC Low Binding Regenerated Cellulose30 KDa mol. wt. cutoff catalog # UFC3LTK00), permeable to the targetinggroup, but not to HSA. A small portion (5-10%) of the sample volume isfiltered by centrifugation at 2000×g for 20 min through the cutofffilter, and the concentration of unbound targeting group in the sampleis measured in the filtrate.

For measuring binding to fibrin, a fibrin clot may be formed in a wellof a microtiter plate and contacted with the targeting group. After anincubation time sufficient to establish equilibrium, the supematant isremoved by aspiration (the insoluble fibrin remains bound as a gelledclot to the bottom of the well). The concentration of unbound targetinggroup in the supernatant is then measured.

In both methodologies, the concentration of bound contrast agent isdetermined as the difference between the total targeting groupconcentration initially present and the unbound targeting groupconcentration following the binding assay. The bound fraction is theconcentration of bound targeting group divided by the concentration oftotal targeting group.

Affinity of contrast agents to a soluble fibrin DD(E) fragment wasexamined as set forth above and is reported in Table 4. The compoundnumbers provided in Table 4 refer to the structures set forth in thedetailed description. This data in multiple determinations has an errorfrequency of no more than 20% in this biological assay.

TABLE 4 Affinity of Compounds for Fibrin Compound Kd, DD(E), μM 4 33 512 6 13 7 12 12 5.0 13 5.1 14 4.9 32 4.7 15 4.0 16 3.5 17 6.2 18 5.9 88.6 9 6.1 10 9.6 11 13 27 0.7 28 5.3 29 0.8 30 3.7 19 10 20 1.2 21 9.122 3.5 31 0.8 23 0.25 24 0.7 25 5.6 42 0.07 43 0.08 44 0.09 45 0.1 330.1 35 0.11 46 0.15 47 0.18 48 0.199 49 0.22 50 0.3 34 0.39

Example 9 Stability of Contrast Agents

Stability was assayed using rat liver homogenate, which contains bothintra- and extracellular enzymes and represents a particularly harshchemical environment for peptide bonds. Freshly prepared rat liverhomogenate (630 μL) was placed in a glass test tube and incubated at 37°C. in a water bath for 4 minutes. To the rat liver homogenate at 37° C.was added 70 μL of a 1 mM solution of test compound. At time points 0,5, 15, 30, and 60 minutes, a 100 μL aliquot of the reaction mixture wasremoved and mixed with 100 μL of methanol in a microfuge tube to quenchthe reaction. The quenched reaction mixture was centrifuged for 3minutes at 10,000 rpm to pellet the precipitated protein. Thesupernatant was analyzed by LC-MS to quantitate the amount of testcompound remaining by comparing the area of the single ion MS peak tothat of a series of standards. Half life (T1/2) was determined byplotting log percent signal remaining vs. time. The data were fit usingan exponential curve fit, wherein T1/2=In2/slope.

The stability data of the below compounds was tested. Compound 5, whichhas chelates at both the C- and N-termini of the peptide, had a dramaticincrease in half-life resulting from resistance to exopeptidasehydrolysis. A comparable increase in half-life was not achieved throughmodification of one terminus with chelates, even when the other terminuswas capped with an unnatural organic moiety as in, for example, compound3 (containing a biphenyl group on the N-terminus).

Structure 1: half-life=<2 min, free N- and amidated C-terminus

Structure 2: half-life=10 min, N-terminus conjugated to chelates andamidated C-terminus.

Structure 3: half-life=9 min, C-terminus conjugated to chelates andN-terminus acylated with para-(phenyl)benzoic acid.

Structure 5: half-life=65 min, both N- and C-termini conjugated tochelates.

Surprisingly, the above data illustrate that the half-life of a peptideis most significantly increased by the addition of gadolinium chelatesto both the C- and the N-termini.

Example 10 Relaxivity of Contrast Agents

The MRI contrast agents of the present invention were evaluated forrelaxivity using a Bruker NMS-120 Minispec NMR spectrometer operating at0.47 Tesla (20 MHz H-1 Larmor frequency) and 37° C. or a Konig-Brownrelaxometer (20 MHz, H-1 Larmor frequency) operating at 35° C. T1 ofwater protons was determined by an inversion recovery pulse sequenceusing the instrument's software. Relaxivity was determined by measuringthe T1 of multiple solutions of the target (for example, homodispersegels of freshly polymerized fibrinogen, 10 mg/mL) containing zero, 20,30, and 40 μM Gd(III), respectively. The samples are incubated at 37° C.for at least 15 minutes to ensure temperature equilibration before theT1 measurement is performed. The Gd(III) content of the samples isdetermined by inductively coupled plasma—mass spectrometry (ICP-MS). Therelaxivity (per Gd(III) ion) is determined by plotting the relaxationrate (1/T1) in s⁻¹ versus the Gd(III) concentration in mM. The slope ofa linear fit to the data gives the relaxivity. The relaxivity of thecompounds in the absence of target is also determined in an analogousmanner, except there is no target present.

Compounds of the invention show increased relaxivity upon binding tofibrin (FIG. 2) as compared with the relaxivity in the absence ofbiological target.

Example 11 Clot Uptake of Contrast Agents

The uptake of a contrast agent into a thrombus (blood clot) wasdetermined by the following method: A 600 g guinea pig (Hartley male)was anaesthetized. An incision was made in the abdomen and the inferiorvena cava (IVC) isolated and the vessel was allowed to recover for 10minutes. A 1 cm portion of the IVC was clamped and human thrombin (50μL, 4 units) was injected into the vessel to promote thrombus formation.The lower clamp was opened and closed allowing partial blood flow to thesegment. After 2-3 minutes the clips were removed. The thrombus wasallowed to age in the animal for 30 minutes. At this point the contrastagent, compound 32 at a dose of 2 μmol/kg and trace radiolabeled with 70μCi of ¹¹¹In, was injected via the jugular vein. Immediately followinginjection of agent compound 32, a non-specific control comprisingGd(DTPA) at a dose of 2 μmol/kg mixed with 70 μCi ^(99m)TcDTPA wasinjected via the jugular vein. After 30 minutes blood was drawn, theanimal sacrificed, and the thrombus removed. The blood sample wasweighed and counted using a Packard Cobra II gamma counter. The thrombuswas also weighed and counted. Counts arising from ^(99m)Tc were detectedfrom 128-165 keV while counts arising from the decay of ¹¹¹In weredetected from 390-500 keV. Control experiments with only ^(99m)Tc or¹¹¹In demonstrated radioactivity arising from ^(99m)Tc was negligible atdetection energies used for ¹¹¹In and vice versa. The radioactive decaydata were converted to % initial dose per gram of tissue, % ID/g, andthe mean of three experiments is presented graphically. Radiolabelingwith ¹¹¹In was performed in advance: An appropriate radiochemical amountof ¹¹¹InCl₃ (New England Nuclear) was added to the fibrin targetedcontrast agent. The pH was adjusted to 4 by addition of 1 M HCl. Thesample was heated at 45° C. for 1 hour. The pH was adjusted to neutralby addition of 1 M NaOH. The labeled agent compound 32 was >95% pure byγ-detected HPLC.

Fibrin-specific agents show a marked increase in clot uptake. FIG. 3shows that an agent, compound 32, is accumulating in the thrombus. Thereis specific clot uptake compared to ^(99m)TcDTPA and there is a higherconcentration of the agent in the thrombus than in the surroundingblood.

Specificity of clot uptake also can be demonstrated using MRI. Theprocedure for in vivo imaging of a thrombus with an agent is as follows:A 600 g guinea pig (Hartley male) is anaesthetized. An incision is madein the throat and one of the jugular veins isolated. A 1 cm section ofthe jugular vein is isolated with vascular clamps. Freshly drawn bloodfrom the animal (50 μL) is mixed with human thrombin (50 μL, 4 units)and is injected into the clamped segment of the vein. Four minutes afterinjection, the clamps are removed and the thrombus is allowed to age for30 minutes. Agent, compound 32, is injected at a dose of 6 μmol/kg andthe throat area of the guinea pig is imaged at 1.5 T using a spoiledgradient method TR=36, TE=5, flip angle=30°. The thrombus appears brightrelative to the blood.

Example 12 Obtaining an MR Image of a Thrombus With a Targeted ContrastAgent with and Without Black Blood

A 2.5 kg female New Zealand White rabbit was anesthetized with acocktail of Ketamine (50 mg/kg), Aceapromazine (2.5 mg/kg), and Rompon(5 mg/kg) and anesthesia maintained with sodium pentobarbital (approx.35 mg/kg as needed). An i.v. catheter (24 g) was placed into the earvein and the ear artery. The jugular vein and carotid artery wereisolated. A stenosis was created in the carotid artery by placing an 18g needle on top of the vessel and then suturing it into place with 3-0suture. The needle was then removed. A 5 mm portion of the artery wasthen segmented off distally to the stenosis with microvascular clips.The artery was then crushed twice along the 5 mm section. The proximalvascular clip was released to allow blood flow into the section for ca.3 sec. The clip was reapplied and artery was crushed twice again alongthe 5 mm section. After 4 minutes, the clips were removed. A 5 mmsegment of the jugular vein was isolated with microvascular clips. Athrombus was created by injecting 100 μL of a 3.7 units of thrombin,0.06 M CaCl₂, rabbit whole blood mixture. After 4 minutes, the clipswere removed.

The thrombi were allowed to age for 50 minutes. A 1.0 mL solution of thethrombus targeted agent (Structure III, 5 mM, 2 μmol/kg) wasadministered via the ear vein. After 10 minutes, the animal was placedinside a General Electric Signa LxCVi 1.5 tesla scanner and a first MRIdata set was obtained using a 3D RF spoiled gradient echo sequence(SPGR: TR=39 ms, TE=3.1 ms, flip angle=40 degrees, field of view=8 cm,acquisition bandwith=31.25 kH). Chemical fat saturation was applied aswell as 40 mm spatial inferior and superior saturation bands.Immediately following this scan (8 minutes later), a second MRI data setwas acquired using the same parameters with the addition of 40 mmspatial inferior and superior saturation bands to generate a “blackblood” image.

FIG. 4A shows the maximum intensity projection (MIP) of the first dataset. The blood vessels are partially enhanced from time of flighteffects. FIG. 4B shows the MIP of the second data set where the signalfrom in-flowing blood was suppressed (black blood) by the use ofsuperior and inferior saturation bands. In FIGS. 4A and 4B, theidentification of the stationary target (a thrombus) through use of thetargeted contrast agent is clearly facilitated.

Example 13 Synthesis of an Optical Contrast Agent

NovaSyn TGR resin (0.20 mmol/g, 100 mg, 20 μmol) was washed withNMP/ether/NMP. The peptide was assembled by the standard solid phasemethod using the PyBOP/HOBt/DIEA activation. After the coupling of thefinal amino acid residue, the resin bound peptide was treated with asolution of piperidine in DMF (20% by volume, 2.0 mL) for 10 minutes toremove the Fmoc protecting group. The resin was washed thoroughly withNMP/ether/NMP, and was treated with a solution offluorascein-5-isothiocyanate (23.4 mg, 60 μmol) andduisopropylethylamine (11.6 mg, 15.7 μL, 90 μmol) in DMF (1.5 mL) for 12hours. The resin was washed thoroughly (NMP/ether/NMP), and treated witha solution of Tl (TFA)₃ (18.7 mg, 34.5 μmol) in DMF (1.5 mL) at 4° C.for three hours. The resin was washed after this treatment, and treatedwith a cocktail of TFA/TIS/water (95/2.5/2.5, 2.0 mL) for two hours. Thecrude peptide was precipitated by adding ether to the cleavage cocktail,and purified by preparative HPLC using a Vydac C-18 column. StructuresA-N were formed in this manner and their fibrin DD(E) fragmentaffinities were determined (Table 5).

TABLE 5 Affinity of optical targeted contrast agents to fibrin. Kd (μM)ID Vs. DD(E) Fluor X₁ X₂ X₃ X₄ X₅ X₆ X₇ X₈ X₉ X₁₀ X₁₁ X₁₂ X₁₃ D 0.061Fluor Ahx W F H C Hyp Y(3-I) D L C H I J 0.055 Fluor Q W E C P Y G L C WI Q K 0.06 Fluor W F H C P Y D L C H I L E 0.09 Fluor Ahx G G F H C HypY(3-I) D L C H I C 0.097 Fluor Ahx G F H C Hyp Y(3-I) D L C H I B 0.099Fluor Ahx F H C Hyp Y(3-I) D L C H I H 0.119 Fluor K W F H C Hyp Y(3-I)D L C H I G 0.124 Fluor K G F H C Hyp Y(3-I) D L C H I I 0.127 Fluor K GG F H C Hyp Y(3-I) D L C H I A 0.136 Fluor beta-A F H C Hyp Y(3-I) D L CH I F 0.212 Fluor K F H C Hyp Y(3-I) D L C H I

N-terminal labeling of the peptides with optical probes can modulate thebinding affinity of the optical contrast agents. For example, whencomparing fluorescein, 4-methoxycoumarin and tetramethylrhodaminederivatives of the peptide (QWECPYGLCWIQ (SEQ ID NO:27); Kd=3 μM) thefollowing Kd's were observed (Table 6):

TABLE 6 Compound Fluorophore Kd J Fluorescein 0.06 LTetramethylrhodamine 2.0 N 4-Methoxycoumarin 0.2

Example 14 Fibrin Targeted Urokinase

Fibrin targeted urokinase is prepared according to the followingprocedure. A fibrin binding peptide with a Gly-Gly dipeptide linker isprepared according to solid phase procedures. The N-terminus of thepeptide is blocked with an acetyl group, and the C-terminal carboxylicacid is converted to a succinamidal active ester. Direct chemicalligation is achieved by mixing urokinase and the activated peptide inappropriate proportions in an aqueous buffer and gently agitating thesolution for 30 minutes.

Fibrin targeted urokinase can be purified by HPLC. Binding to fibrin canbe assessed. Compound 1 binds fibrin selectively versus fibrinogen.

The rabbit jugular vein model of Collen et al. (J. Clin. Invest. 1983,71, 368-376) is used for thrombolysis assays. Compound (2 mg/kg) isadministered by infusion of a bolus (consisting of 20% of the totaldose) over 1 min, along with a heparin bolus (300 units/kg) over 1 min.The remainder of the dose is continuously infused over the next 60 min,and heparin (60 units/kg/hr) is continuously infused over the next 180min. At 3 hours, the animals are sacrificed, and clots analyzed.Compound 1 is more potent in clot lysis than scuPA alone. At 3 hr, with2 mg/kg of compound 1, there is less consumption of fibrinogen andα2-antiplasmin, relative to equivalent doses of scuPA alone,demonstrating that compound 1 was more fibrin specific than scuPA alone.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A contrast agent, or a pharmaceutically acceptable salt thereof,comprising a peptide, said peptide modified at its N- and its C-termini,independently, with a moiety comprising one or more metal chelatecomplexes, wherein said contrast agent has the formula:

wherein: Chelate represents a metal chelate complex; Linker represents alinker moiety; Linker-subunit represents a linker-subunit moiety; m isindependently an integer from 1 to 10; p is independently an integerfrom 0 to 5; s is independently 0 or 1; n is an integer from 3 to 50,inclusive; R¹ is an amino acid side chain or a non-natural amino acidside chain; and R² is independently a hydrogen or an aliphatic group. 2.The contrast agent of claim 1, the contrast agent having a structureselected from the group consisting of:

wherein Gd is a paramagnetic metal ion Gd(III), and wherein the Gd(III)is coordinated to the DTPA moiety; and wherein the DTPA moiety iscovalently linked to a moiety comprising a C(═O) group at an ethylene oracetate carbon on the DTPA moiety.
 3. The contrast agent of claim 1, thecontrast agent having a structure selected from the group consisting of:


4. The contrast agent of claim 1, the contrast agent having a structureselected from the group consisting of:


5. A pharmaceutically acceptable salt of a contrast agent of claims 1-4,wherein said salt is a counter ion of organic or inorganic acids orbases, or mixtures thereof.
 6. A pharmaceutically acceptable salt of acontrast agent of claims 1-4, wherein said salt is a sodium salt.
 7. Apharmaceutically acceptable salt of a contrast agent, said contrastagent having the structure:


8. The pharmaceutically acceptable salt of claim 7, wherein said salt isa sodium salt.
 9. A pharmaceutically acceptable salt of a contrastagent, said contrast agent having the structure:


10. The pharmaceutically acceptable salt of claim 9, wherein said saltis a sodium salt.